Chlorophyll fluorescence emission as a reporter on cold tolerance in Arabidopsis thaliana accessions

Non-invasive, high-throughput screening methods are valuable tools in breeding for abiotic stress tolerance in plants. Optical signals such as chlorophyll fluorescence emission can be instrumental in developing new screening techniques. In order to examine the potential of chlorophyll fluorescence to reveal plant tolerance to low temperatures, we used a collection of nine Arabidopsis thaliana accessions and compared their fluorescence features with cold tolerance quantified by the well established electrolyte leakage method on detached leaves. We found that, during progressive cooling, the minimal chlorophyll fluorescence emission rose strongly and that this rise was highly dependent on the cold tolerance of the accessions. Maximum quantum yield of PSII photochemistry and steady state fluorescence normalized to minimal fluorescence were also highly correlated to the cold tolerance measured by the electrolyte leakage method. In order to further increase the capacity of the fluorescence detection to reveal the low temperature tolerance, we applied combinatorial imaging that employs plant classification based on multiple fluorescence features. We found that this method, by including the resolving power of several fluorescence features, can be well employed to detect cold tolerance already at mild sub-zero temperatures. Therefore, there is no need to freeze the screened plants to the largely damaging temperatures of around −15°C. This, together with the method''s easy applicability, represents a major advantage of the fluorescence technique over the conventional electrolyte leakage method.Key words: chlorophyll fluorescence, cold acclimation, electrolyte leakage, high-throughput screening, natural accessions     

Antibiotic utilization and Pseudomonas aeruginosa resistance in intensive care units

Pseudomonas aeruginosa is one of the most frequent and dangerous pathogens involved in the etiology of severe nosocomial infections. A retrospective observational study was conducted at all intensive care units of the University Hospital in Olomouc, Czech Republic (155 ICU beds). Complete antibiotic utilization data of the ICUs in the period of 1999 to 2008 were processed according to ATC/DDD system and expressed in defined daily doses per 100 bed-days (DBD). Utilization of meropenem, imipenem, ciprofloxacin, ofloxacin, pefloxacin, gentamicin, amikacin, ceftazidime, cefoperazone, cefoperazone/sulbactam and piperacillin/tazobactam was measured. Pseudomonas aeruginosa strains were isolated from clinical material obtained from patients hospitalized in ICUs. During the ten-year period, utilization of the entire group of antibiotics monitored grew. It increased from 23.52 DBD in 1999 to 27.48 DBD in 2008 with a peak of 33.04 DBD in 2007. P. aeruginosa accounted for as much as 42% of pneumonias and 23% of surgical wound infections. Our results show that P. aeruginosa strains became gradually resistant to all antibiotics used in the treatment of the infections caused by them, with the exception of amikacin and piperacillin/tazobactam.     

Selective BRAFV600E inhibitor PLX4720, requires TRAIL assistance to overcome oncogenic PIK3CA resistance

Documented sensitivity of melanoma cells to PLX4720, a selective BRAFV600E inhibitor, is based on the presence of mutant BRAF(V600E) alone, while wt-BRAF or mutated KRAS result in cell proliferation. In colon cancer appearance of oncogenic alterations is complex , since BRAF, like KRAS mutations, tend to co-exist with those in PIK3CA and mutated PI3K has been shown to interfere with the successful application of MEK inhibitors. When PLX4720 was used to treat colon tumours, results were not encouraging and herein we attempt to understand the cause of this recorded resistance and discover rational therapeutic combinations to resensitize oncogene driven tumours to apoptosis. Treatment of two genetically different BRAF(V600E) mutant colon cancer cell lines with PLX4720 conferred complete resistance to cell death. Even though p-MAPK/ ERK kinase (MEK) suppression was achieved, TRAIL, an apoptosis inducing agent, was used synergistically in order to achieve cell death by apoptosis in RKO(BRAFV600E/PIK3CAH1047) cells. In contrast, for the same level of apoptosis in HT29(BRAFV600E/PIK3CAP449T) cells, TRAIL was combined with 17-AAG, an Hsp90 inhibitor. For cells where PLX4720 was completely ineffective, 17-AAG was alternatively used to target mutant BRAF(V600E). TRAIL dependence on the constitutive activation of BRAF(V600E) is emphasised through the overexpression of BRAF(V600E) in the permissive genetic background of colon adenocarcinoma Caco-2 cells. Pharmacological suppression of the PI3K pathway further enhances the synergistic effect between TRAIL and PLX4720 in RKO cells, indicating the presence of PIK3CA(MT) as the inhibitory factor. Another rational combination includes 17-AAG synergism with TRAIL in a BRAF(V600E) mutant dependent manner to commit cells to apoptosis, through DR5 and the amplification of the apoptotic pathway. We have successfully utilised combinations of two chemically unrelated BRAF(V600E) inhibitors in combination with TRAIL in a BRAF(V600E) mutated background and provided insight for new anti-cancer strategies where the activated PI3KCA mutation oncogene should be suppressed.     

Genetic differentiation of the Western Capercaillie highlights the importance of South-eastern Europe for understanding the species phylogeography

The Western Capercaillie (Tetrao urogallus L.) is a grouse species of open boreal or high altitude forests of Eurasia. It is endangered throughout most mountain range habitat areas in Europe. Two major genetically identifiable lineages of Western Capercaillie have been described to date: the southern lineage at the species' southernmost range of distribution in Europe, and the boreal lineage. We address the question of genetic differentiation of capercaillie populations from the Rhodope and Rila Mountains in Bulgaria, across the Dinaric Mountains to the Slovenian Alps. The two lineages' contact zone and resulting conservation strategies in this so-far understudied area of distribution have not been previously determined. The results of analysis of mitochondrial DNA control region sequences of 319 samples from the studied populations show that Alpine populations were composed exclusively of boreal lineage; Dinaric populations of both, but predominantly (96%) of boreal lineage; and Rhodope-Rila populations predominantly (>90%) of southern lineage individuals. The Bulgarian mountains were identified as the core area of the southern lineage, and the Dinaric Mountains as the western contact zone between both lineages in the Balkans. Bulgarian populations appeared genetically distinct from Alpine and Dinaric populations and exhibited characteristics of a long-term stationary population, suggesting that they should be considered as a glacial relict and probably a distinct subspecies. Although all of the studied populations suffered a decline in the past, the significantly lower level of genetic diversity when compared with the neighbouring Alpine and Bulgarian populations suggests that the isolated Dinaric capercaillie is particularly vulnerable to continuing population decline. The results are discussed in the context of conservation of the species in the Balkans, its principal threats and legal protection status. Potential conservation strategies should consider the existence of the two lineages and their vulnerable Dinaric contact zone and support the specificities of the populations.     

Comparison of genetic algorithms for experimental multi-objective optimization on the example of medium design for cyanobacteria

In this work, two different genetic algorithms were applied to improve culture media composition for the autotrophic cyanobacteria Synechococcus PCC 7942. Biomass yield and conversion of the asymmetric reduction of 2', 3', 4', 5', 6'-pentafluoroacetophenone were considered as simultaneous objectives, resulting in a multi-objective optimization problem. Even when similar performances of both algorithms were observed, it could be shown that a novel strength pareto approach was able to achieve remarkable results with a reduced number of experiments (160 instead of 320). Handling a high number of media components (13), their concentrations were adjusted, delivering high improvements in comparison to the standard BG 11 culture media. The quality of the Synechococcus biocatalyst could be increased up to fivefold compared to the initial state of the optimization.     

Adiponectin and negative mood in healthy premenopausal and postmenopausal women

Negative mood and stress are associated with cardiovascular and metabolic disease. There are likely many physiological mechanisms underlying the poor health outcomes. The relationship of psychological states (negative mood, life stress, and stress-responsive hormones) and adiponectin, an adipokine that promotes insulin sensitivity, was investigated in two separate studies. The two groups of participants included 52 healthy, premenopausal women, and 63 postmenopausal women with a range of stress levels. The relationship between adiponectin and psychological state (perceived stress and negative mood) was examined cross-sectionally in both groups of participants, but also prospectively (1 year later) in the group of postmenopausal women.In premenopausal women, negative mood and nocturnal urinary epinephrine were significantly related to adiponectin, independent of BMI. In postmenopausal women, negative mood was not associated with adiponectin cross-sectionally, but negative mood was a significant predictor for lower levels of adiponectin 1 year later, independent of initial adiponectin concentrations and changes in body mass index. Lastly, having a depressive disorder was related to lower adiponectin. As adiponectin levels are associated with insulin resistance, obesity, and diabetes mellitus, these findings suggest there may be an adiponectin-mediated pathway explaining in part how negative mood affects metabolic health. Mechanistic studies are needed to explore this potential relationship further.     

Cyanobacterial small chlorophyll-binding protein ScpD (HliB) is located on the periphery of photosystem II in the vicinity of PsbH and CP47 subunits

Cyanobacteria contain several genes coding for small one-helix proteins called SCPs or HLIPs with significant sequence similarity to chlorophyll a/b-binding proteins. To localize one of these proteins, ScpD, in the cells of the cyanobacterium Synechocystis sp. PCC 6803, we constructed several mutants in which ScpD was expressed as a His-tagged protein (ScpDHis). Using two-dimensional native-SDS electrophoresis of thylakoid membranes or isolated Photosystem II (PSII), we determined that after high-light treatment most of the ScpDHis protein in a cell is associated with PSII. The ScpDHis protein was present in both monomeric and dimeric PSII core complexes and also in the core subcomplex lacking CP43. However, the association with PSII was abolished in the mutant lacking the PSII subunit PsbH. In a PSII mutant lacking cytochrome b(559), which does not accumulate PSII, ScpDHis is associated with CP47. The interaction of ScpDHis with PsbH and CP47 was further confirmed by electron microscopy of PSII labeled with Ni-NTA Nanogold. Single particle image analysis identified the location of the labeled ScpDHis at the periphery of the PSII core complex in the vicinity of the PsbH and CP47. Because of the fact that ScpDHis did not form any large structures bound to PSII and because of its accumulation in PSII subcomplexes containing CP47 and PsbH we suggest that ScpD is involved in a process of PSII assembly/repair during the turnover of pigment-binding proteins, particularly CP47.