Neuronal plasticity in the mushroom body calyx during adult maturation in the honeybee and possible pheromonal influences

Honeybee workers express a pronounced age‐dependent polyethism switching from various indoor duties to foraging outside the hive. This transition is accompanied by tremendous changes in the sensory environment that sensory systems and higher brain centers have to cope with. Foraging and age have earlier been shown to be associated with volume changes in the mushroom bodies (MBs). Using age‐ and task‐controlled bees this study provides a detailed framework of neuronal maturation processes in the MB calyx during the course of natural behavioral maturation. We show that the MB calyx volume already increases during the first week of adult life. This process is mainly driven by broadening of the Kenyon cell dendritic branching pattern and then followed by pruning of projection neuron axonal boutons during the actual transition from indoor to outdoor duties. To further investigate the flexible regulation of division of labor and its neuronal correlates in a honeybee colony, we studied the modulation of the nurse‐forager transition via a chemical communication system, the primer pheromone ethyl oleate (EO). EO is found at high concentrations on foragers in contrast to nurse bees and was shown to delay the onset of foraging. In this study, EO effects on colony behavior were not as robust as expected, and we found no direct correlation between EO treatment and synaptic maturation in the MB calyx. In general, we assume that the primer pheromone EO rather acts in concert with other factors influencing the onset of foraging with its effect being highly adaptive. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1368–1384, 2015     

General method of rapid Smith/Birnstiel mapping adds for gap closure in shotgun microbial genome sequencing projects: application to Pseudomonas putida KT2440

A physical mapping strategy has been developed to verify and accelerate the assembly and gap closure phase of a microbial genome shotgun-sequencing project. The protocol was worked out during the ongoing Pseudomonas putida KT2440 genome project. A macro-restriction map was constructed by linking probe hybridisation of SwaI- or I-CeuI-restricted chromosomes to serve as a backbone for the quick quality control of sequence and contig assemblies. The library of PCR-generated SwaI linking probes was derived from the sequence assembly after 3- and 6-fold genome coverage. In order to support gap closure in regions with ambiguous assemblies such as the repetitive sequence of the seven ribosomal operons, high-resolution Smith/Birnstiel maps were generated by Southern hybridisation of pulsed-field gel electrophoresis-separated rare-cutter complete/frequent-cutter partial digestions with rare-cutter fragment end probes. Overall 1.5 Mb of the 6.1 Mb P.putida KT2440 genome has been subjected to high-resolution physical mapping in order to align assemblies generated from shotgun sequencing.     

Properties of an LNA-modified ricin RNA aptamer

'Locked nucleic acids' (LNAs) are sugar modified nucleic acids containing the 2'-O-4'C-methylene-β-D-ribofuranoses. The substitution of RNAs with LNAs leads to an enhanced thermostability. Aptamers are nucleic acids, which are selected for specific target binding from a large library pool by the 'SELEX' method. Introduction of modified nucleic acids into aptamers can improve their stability. The stem region of a ricin A chain RNA aptamer was substituted by locked nucleic acids. Different constructs of the LNA-substituted aptamers were examined for their thermostability, binding activity, folding and RNase sensitivity as compared to the natural RNA counterpart. The LNA-modified aptamers were active in target binding, while the loop regions and the adjacent stem nucleotides remained unsubstituted. The thermostability and RNase resistance of LNA substituted aptamers were enhanced as compared to the native RNA aptamer. This study supports the approach to substitute the aptamer stem region by LNAs and to leave the loop region unmodified, which is responsible for ligand binding. Thus, LNAs possess an encouraging potential for the development of new stabilized nucleic acids and will promote future diagnostic and therapeutic applications.     

High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase

The BTL2 lipase gene from Bacillus thermocatenulatus was subcloned into the pPICZalphaA vector and integrated further into the genome of Pichia pastoris GS115. One of the best transformants harboring the linearized plasmid pPalpha-BTL2 integrating into the P. pastoris genomic DNA was cultivated in a 5-L bioreactor filled with 4L of the culture medium BMMY. The BTL2 lipase was produced as an extracellular protein in large quantities of 309,000U/L supernatant. The lipase was purified using butyl-Sepharose with a specific activity of 23,000U/mg protein towards tributyrin. The pure enzyme was characterized and its physicochemical properties were compared to those of the BTL2 lipase, which had previously been expressed in Escherichia coli under the control of its native promoter on pUC18 or under the control of the strong temperature inducible promoter lambdaP(L), yielding 600U/g or 54,000U/g wet cells, respectively. The three proteins showed the same N-terminal sequence and had very similar pH optimum, pH stability, temperature optimum, thermostability, and substrate specificity profiles. Three enzymes were extremely stable in the presence of several organic solvents and detergents.     

Chaperone mediated autophagy contributes to the newly synthesized histones H3 and H4 quality control

Although there are several pathways to ensure that proteins are folded properly in the cell, little is known about the molecular mechanisms regulating histone folding and proteostasis. In this work, we identified that chaperone-mediated autophagy (CMA) is the main pathway involved in the degradation of newly synthesized histones H3 and H4. This degradation is finely regulated by the interplay between HSC70 and tNASP, two histone interacting proteins. tNASP stabilizes histone H3 levels by blocking the direct transport of histone H3 into lysosomes. We further demonstrate that CMA degrades unfolded histone H3. Thus, we reveal that CMA is the main degradation pathway involved in the quality control of histone biogenesis, evidencing an additional mechanism in the intricate network of histone cellular proteostasis.     

Expressed antibody repertoires in human cord blood cells: 454 sequencing and IMGT/HighV-QUEST analysis of germline gene usage, junctional diversity, and somatic mutations

Human cord blood cell-derived IgM antibodies are important for the neonate immune responses and construction of germline-based immunoglobulin libraries. Several previous studies of a relatively small number of sequences found that they exhibit restrictions in the usage of germline genes and in the diversity of the variable heavy chain complementarity determining region 3 compared to adults. To further characterize such restrictions on a larger scale and to compare the early B-cell diversity to adult IgM repertoires, we performed 454 sequencing and IMGT/HighV-QUEST analysis of cord blood IG libraries from two babies and determined germline gene usage, V-D-J rearrangement, VHCDR3 diversity, and somatic mutations to characterize human neonate repertoire. Most of the germline subgroups were identified with frequencies comparable to those present in the adult IgM repertoire except for the IGHV1-2 gene that was preferentially expressed in the cord blood cells. The gene usage diversity contributed to 1,430 unique IGH V-D-J rearrangement patterns while the exonuclease trimming and N region addition at the V-D-J junctions along with gene diversity created a wide range of VHCDR3 with different lengths and sequence variability. We observed a lower degree of somatic mutations in the CDR and framework regions of antibodies from cord blood cells compared to adults. These results provide insights into the characteristics of human cord blood antibody repertoires, which have gene usage diversity and VHCDR3 lengths similar to that of the adult IgM repertoire but differ significantly in some of the gene usages, V-D-J rearrangements, junctional diversity, and somatic mutations.     

Influence of habitat complexity and landscape configuration on pollination and seed-dispersal interactions of wild cherry trees

Land-use intensification is a major cause for the decline in species diversity in human-modified landscapes. The loss of functionally important species can reduce a variety of ecosystem functions, such as pollination and seed dispersal, but the intricate relationships between land-use intensity, biodiversity and ecosystem functioning are still contentious. Along a gradient from forest to intensively used farmland, we quantified bee species richness, visitation rates of bees and pollination success of wild cherry trees (Prunus avium). We analysed the effects of structural habitat diversity at a local scale and of the proportion of suitable habitat around each tree at a landscape scale. We compared these findings with those from previous studies of seed-dispersing birds and mammals in the same model system and along the same land-use gradient. Bee species richness and visitation rates were found to be highest in structurally simple habitats, whereas bird species richness—but not their visitation rates—were highest in structurally complex habitats. Mammal visitation rates were only influenced at the landscape scale. These results show that different functional groups of animals respond idiosyncratically to gradients in habitat and landscape structure. Despite strong effects on bees and birds, pollination success and bird seed removal did not differ along the land-use gradient at both spatial scales. These results suggest that mobile organisms, such as bees and birds, move over long distances in intensively used landscapes and thereby buffer pollination and seed-dispersal interactions. We conclude that measures of species richness and interaction frequencies are not sufficient on their own to understand the ultimate consequences of land-use intensification on ecosystem functioning.     

The role of fluoride on the process of titanium corrosion in oral cavity

Titanium is known to possess excellent biocompatibility as a result of corrosion resistance, lack of allergenicity when compared with many other metals. Fluoride is well known as a specific and effective caries prophylactic agent and its systemic application has been recommended widely over recent decades. Nevertheless, high fluoride concentrations impair the corrosion resistance of titanium. The purpose of this article is to summarize the current data regarding the influence of fluoride on titanium corrosion process in the last 5?years. These data demonstrate noxious effects induced by high fluoride concentration as well as low pH in the oral cavity. Therefore, such conditions should be considered when prophylactic actions are administrated in patients containing titanium implants or other dental devices.