Affinity resins containing enzymatically resistant mRNA cap analogs--a new tool for the analysis of cap-binding proteins

Cap-binding proteins have been routinely isolated using m⁷GTP-Sepharose; however, this resin is inefficient for proteins such as DcpS (scavenger decapping enzyme), which interacts not only with the 7-methylguanosine, but also with the second cap base. In addition, DcpS purification may be hindered by the reduced resin capacity due to the ability of DcpS to hydrolyze m⁷GTP. Here, we report the synthesis of new affinity resins, m⁷GpCH₂pp- and m⁷GpCH₂ppA-Sepharoses, with attached cap analogs resistant to hydrolysis by DcpS. Biochemical tests showed that these matrices, as well as a hydrolyzable m⁷GpppA-Sepharose, bind recombinant mouse eIF4E^(28-217) specifically and at high capacity. In addition, purification of cap-binding proteins from yeast extracts confirmed the presence of all expected cap-binding proteins, including DcpS in the case of m⁷GpCH₂pp- and m⁷GpCH₂ppA-Sepharoses. In contrast, binding studies in vitro demonstrated that recombinant human DcpS efficiently bound only m⁷GpCH₂ppA-Sepharose. Our data prove the applicability of these novel resins, especially m⁷GpCH₂ppA-Sepharose, in biochemical studies such as the isolation and identification of cap-binding proteins from different organisms.     

RAD sequencing,morphometry and synecology clarify the taxonomy of the Melica ciliata (Poaceae) complex in France and Poland

Melica (Poaceae) consist of about 92 species distributed across temperate regions of the world. Within section Dalycum, Melica ciliata sensu lato forms a taxonomic complex of several species and subspecies with clinal morphological variation causing conflicting identifications. To resolve taxonomic confusion, we used three complementary approaches, through molecular, morphological, and phytoecological analyses. The double-digest restriction-associated DNA markers significantly support the monophyly of three taxa: (i) the Mediterranean Melica magnolii, (ii) the Eurasian Melica transsilvanica subsp. transsilvanica, and (iii) the west-European M. ciliata subsp. glauca. This differentiation is corroborated by the analysis of 22 morphometric variables. Furthermore, phytoecological analysis of 221 floristic inventories revealed habitat distinctions among these taxa. Our approach of integrative taxonomy argues for a specific distinction for these three taxa, and we include a key to separate these forms. These new molecular data on the section Dalycum, subsection Ciliatae, call for further phylogenetic analyses including samples of M. ciliata subsp. ciliata and other East-Mediterranean and South African taxa.     

Electrophoretic analysis of RNA from rotavirus strains isolated from children in the years 1986-1990]

Double stranded RNA of rotaviruses is composed of 11 segments which in standard and field strains may differ in electrophoretic mobility in polyacrylamide gel electrophoresis. This study was aimed at determination of electrophoretic differentiation of rotavirus strains found in samples of feces of children treated because of diarrhoea in two hospitals in Warsaw and in Kraków region in the years 1986-1990. Out of 85 positive samples from Warsaw and 90 from Krakow region, for investigation 44 and 36 strains, respectively were included. Totally for 71 strains well stained 11 segments of RNA were obtained which permitted for determination of their electrophoretic-types. Majority of strains, 60 (84%) were characterized by a long electrophoretic-type, whereas 11 (16%) had a short electrophoretic-type. Considering differences in migration speed of 1-4 and 7-9 segments, within long and short types, eight subtypes denominated as a-h were present. Subtypes a, c and d constituted 73% of all tested strains. These studies indicate that in Poland occur at least eight electrophoretic subtypes of rotaviruses and that this feature is partly correlated with belonging of strains to subgroups and antigenic serotypes.     

The synergism of natural compounds in the pursuit of safe and healthier food

Food producers apply modern processing techniques and use a variety of preservative additives to guarantee safe food and a longer shelflife. Regrettably many of these impact the sensory characteristics of the foodstuffs, such as colour, texture, and flavour, which can result in low consumer acceptance. Additionally, strategies used to reduce growth of spoilage and pathogenic bacteria are not selective enough and may inactivate also desired microbiota. Food is usually overdosed with antimicrobials that are supplemented ‘just in case.’ Consequently, food producers are searching for natural preservation methods that are not harmful to humans. Nature offers a wide spectrum of biologically active (phyto) chemicals that can be used as potential natural preservatives. Compounds with bacterial growth-limiting properties are detected in all parts of plants, including their leaves, flowers, fruits, roots, etc. These are mostly acids, alcohols, medium and long-chain organic acids, terpenic compounds, and their derivatives. This study focused on the effectiveness of plant extracts, i.e., synergism between terpenoids and medium chain fatty acids in cured cooked meat. Bacterial strains that were tested include typical members of the spoilage microflora in vacuum (Lactobacillus curvatus) and MA-packed meats (Brochothrix thermosphacta). These were isolated and identified in a separate study. L. curvatus was observed to be very resistant against either terpenoids or fatty acids when used separately, whereas its growth was strongly inhibited when both chemicals were combined. Growth of B. thermosphacta was significantly inhibited when antimicrobial compounds were solely applied, whereas a blend of terpenoids and fatty acids showed an almost bactericidal effect.     

MegaSNPHunter: a learning approach to detect disease predisposition SNPs and high level interactions in genome wide association study

Background  

The interactions of multiple single nucleotide polymorphisms (SNPs) are highly hypothesized to affect an individual's susceptibility to complex diseases. Although many works have been done to identify and quantify the importance of multi-SNP interactions, few of them could handle the genome wide data due to the combinatorial explosive search space and the difficulty to statistically evaluate the high-order interactions given limited samples.     

Diversity of lactic acid bacteria of the bioethanol process

Background  

Bacteria may compete with yeast for nutrients during bioethanol production process, potentially causing economic losses. This is the first study aiming at the quantification and identification of Lactic Acid Bacteria (LAB) present in the bioethanol industrial processes in different distilleries of Brazil.     

Iron-sulfur cluster reconstitution of spinach chloroplast Rieske protein requires a partially prefolded apoprotein

The Rieske 2Fe-2S protein is a central component of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for expression in Escherichia coli of full-length and truncated Spinacia oleracea Rieske (PetC) proteins fused to the MalE, maltose binding protein. The expressed Rieske fusion proteins were found predominantly in soluble form in the E. coli cytoplasm. These proteins could be readily purified for further experimentation. In vitro reconstitution of the characteristic, "Rieske-type" 2Fe-2S cluster into these fused proteins was accomplished by a chemical method employing reduced iron and sulfide. Cluster incorporation was monitored by electron paramagnetic resonance and optical circular dichroism (CD) spectroscopy. CD spectral analysis in the ultraviolet region suggests that the spinach Rieske apoprotein must be in a partially folded conformation to incorporate an appropriate iron-sulfur cluster. These data further suggest that upon cluster integration, further folding occurs, allowing the Rieske protein to attain a final, native structure. The data presented here are the first to demonstrate successful chemical reconstitution of the 2Fe-2S cluster into a Rieske apoprotein from higher plant chloroplasts.     

Linking elements in German Origin, Change, Functionalization

Contemporary German is known for its complex system of linking elements. They not only show different degrees of productivity (between unproductive -es- and very productive -s-), but also exhibit functional diversity, with some of them even allowing plural interpretation, e.g. -er- in Völk+er+kunde ‘ethnology’ vs. Volk+s+kunde ‘folklore’. In this paper, we argue that this is due to the complex historical development from two different sources. The first layer of linking elements, which arose out of Germanic primary suffixes, was reduced to one member, the “older” linking -e-, already in Old High German (e.g. in NHG Tag+e+werk ‘daily task’). The current functional diversity of the linking elements is primarily due to the later evolution out of inflectional endings. The dissociation from the second source has included a gradual change of the assignment rules from lexical (gender, declension class) to prosodic (formal) level. Thus, the current distribution of the most developed linking -s- is the most formalized one, as it can be directly deduced from the prosodic form of the first constituent. The development of the second layer of linking elements resembles the process of grammaticalization. However, linking elements form part of word formation and therefore are not the typical result of grammaticalization.