Guidelines: From artificial evolution to computational evolution: a research agenda

Computational scientists have developed algorithms inspired by natural evolution for at least 50 years. These algorithms solve optimization and design problems by building solutions that are 'more fit' relative to desired properties. However, the basic assumptions of this approach are outdated. We propose a research programme to develop a new field: computational evolution. This approach will produce algorithms that are based on current understanding of molecular and evolutionary biology and could solve previously unimaginable or intractable computational and biological problems.     

Synthesis and biological activity of olomoucine II

Based on our previous experiences with synthesis of purines, novel 2,6,9-trisubstituted purine derivatives were prepared and assayed for the ability to inhibit CDK1/cyclin B kinase. One of newly synthesized compounds designated as olomoucine II, 6-[(2-hydroxybenzyl)amino]-2-[[1-(hydroxymethyl)propyl]amino]-9-isopropylpurine, displays 10 times higher inhibitory activity than roscovitine, potent and specific CDK1 inhibitor. Olomoucine II in vitro cytotoxic activity exceeds purvalanol A, the most potent CDK inhibitor, as it kills the CEM cells with IC(50) value of 3.0 microM.     

Stoichiometry of MutS and MutL at unrepaired mismatches in vivo suggests a mechanism of repair

Mismatch repair (MMR) is an evolutionarily conserved DNA repair system, which corrects mismatched bases arising during DNA replication. MutS recognizes and binds base pair mismatches, while the MutL protein interacts with MutS-mismatch complex and triggers MutH endonuclease activity at a distal-strand discrimination site on the DNA. The mechanism of communication between these two distal sites on the DNA is not known. We used functional fluorescent MMR proteins, MutS and MutL, in order to investigate the formation of the fluorescent MMR protein complexes on mismatches in real-time in growing Escherichia coli cells. We found that MutS and MutL proteins co-localize on unrepaired mismatches to form fluorescent foci. MutL foci were, on average, 2.7 times more intense than the MutS foci co-localized on individual mismatches. A steric block on the DNA provided by the MutHE56A mutant protein, which binds to but does not cut the DNA at the strand discrimination site, decreased MutL foci fluorescence 3-fold. This indicates that MutL accumulates from the mismatch site toward strand discrimination site along the DNA. Our results corroborate the hypothesis postulating that MutL accumulation assures the coordination of the MMR activities between the mismatch and the strand discrimination site.     

Development of arbuscular mycorrhizal biotechnology and industry: current achievements and bottlenecks

Advanced scientific knowledge on arbuscular mycorrhizal symbioses recently enhanced potential for implementation of mycorrhizal biotechnology in horticulture and agriculture plant production, landscaping, phytoremediation and other segments of the plant market. The advances consist in significant findings regarding:—new molecular detection tools for tracing inoculated fungi in the field;—the coexistence mechanisms of various fungi in the single root system;—new knowledge on in vitro physiology of the AM fungi grown in root organ cultures;—mechanisms of synergistic interactions with other microbes like PGPR or saprotrophic fungi; discovery of mycorrhiza supportive compounds such as strigolactones. Scientific knowledge has been followed by technological developments like novel formulations for liquid applications or seed coating, mycorrhiza stimulating compounds or new application modes. Still the missing components of biotechnology are appropriate, cheap, highly reproducible and effective methods for inocula purity testing and quality control. Also there is a weak traceability of the origin of the mycorrhizal fungi strains used in commercial inocula. Numerous poor quality products can still be found on the markets claiming effective formation mycorrhiza which have very low capacity to do so. These products usually rely in their effects on plant growth not on support of host plants via formation of effective mycorrhizal symbiosis but on fertilizing compounds added to products. There is growing number of enterprises producing mycorrhiza based inocula recently not only in developed world but increasingly in emerging markets. Also collaboration between private sector and scientific community has an improving trend as the development of private sector can fuel further research activities. Last but not least there is apparent growing pull of the market and increasing tendency of reduction of agrochemical inputs and employment of alternative strategies in planting and plant production. These circumstances support further developments of mycorrhizal inocula production and applications and maturation of the industry.     

An Effective Combination of Two Different Methods of Postconditioning

Ischemic tolerance based on the synthesis of protective proteins acquires its full strength by repeated exposure to stress, and "the end effector of tolerance" may paradoxically be activated by the second or lethal stress, particularly in the case of preconditioning. That happens when an additional nonspecific stressor is applied either before (preconditioning) or after (postconditioning) the period of lethal ischemia. A combination of antioxidants with pre or postconditioning prevents the acquisition of tolerance, and in the case of more severe attacks repeated stress can lead to accumulation of damage. Our attempt to weaken ischemic injury to hippocampal CA1 with antioxidants applied after lethal stress, i.e. before delayed postconditioning, was ineffective. We then tried using rapid postconditioning consisting of 30-s reperfusion alternating with 15-s ischemia repeated three times and applied immediately at the end of lethal ischemia as a tool decreasing post-ischemic production of reactive oxygen species, and combining that with delayed postconditioning consisting of an i.p. injection of Bradykinin 2?days after lethal ischemia. This approach once more confirmed the efficacy of both rapid as well as delayed postconditioning but, more importantly, it demonstrated the possibility of effectively combining these two procedures. Our findings further confirm that in cases of delayed neuronal death, which is practically pathologically-induced apoptosis, there exists a 2-day-wide therapeutic window that can be effectively exploited.     

TGF-β1 signaling plays a dominant role in the crosstalk between TGF-β1 and the aryl hydrocarbon receptor ligand in prostate epithelial cells

Crosstalk between the aryl hydrocarbon receptor (AhR) and transforming growth factor-β1 (TGF-β1) signaling has been observed in various experimental models. However, both molecular mechanism underlying this crosstalk and tissue-specific context of this interaction are still only partially understood. In a model of human non-tumorigenic prostate epithelial cells BPH-1, derived from the benign prostatic hyperplasia, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) persistently activates the AhR signaling pathway and induces expression of xenobiotic metabolizing enzymes, such as CYP1A1 or CYP1B1. Here we demonstrate that TGF-β1 suppresses the AhR-mediated gene expression through multiple mechanisms, involving inhibition of AhR expression and down-regulation of nuclear AhR, via a SMAD4-dependent pathway. In contrast, TCDD-induced AhR signaling does not affect either TGF-β1-regulated gene expression or epithelial-to-mesenchymal transition. These observations suggest that, in the context of prostate epithelium, TGF-β1 signaling plays a dominant role in the crosstalk with AhR signaling pathway. Given the importance of TGF-β1 signaling in regulation of prostate epithelial tissue homeostasis, as well as the recently revealed role of AhR in prostate development and tumorigenesis, the above findings contribute to our understanding of the mechanisms underlying the crosstalk between the two signaling pathways in the prostate-specific context.     

The Claudin Megatrachea Protein Complex

Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates.     

A new route for chitosan immobilization onto polyethylene surface

Low-density polyethylene (LDPE) belongs to commodity polymer materials applied in biomedical applications due to its favorable mechanical and chemical properties. The main disadvantage of LDPE in biomedical applications is low resistance to bacterial infections. An antibacterial modification of LDPE appears to be a solution to this problem. In this paper, the chitosan and chitosan/pectin multilayer was immobilized via polyacrylic acid (PAA) brushes grafted on the LDPE surface. The grafting was initiated by a low-temperature plasma treatment of the LDPE surface. Surface and adhesive properties of the samples prepared were investigated by surface analysis techniques. An antibacterial effect was confirmed by inhibition zone measurements of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The chitosan treatment of LDPE led to the highest and most clear inhibition zones (35mm(2) for E. coli and 275mm(2) for S. aureus).