Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes

Background  

Recombinant protein production in Escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed.     

Coming to Grips with Evolvability

To explain the evolution of complex organisms by random mutation, drift, and selection is not a trivial task. This becomes obvious if we imagine an organism in which most genes affect most traits and all mutations are immediately expressed in the phenotype. Most of the mutations will be deleterious. Computer programmers experienced a similar problem when trying to evolve computer programs by introducing random changes to a conventional computer code, realizing that almost all random changes are “lethal.” Everyone who has done any programming knows that conventional computer languages are very brittle! Real organisms are not organized in this way but rather involve mediation between the genes and the phenotypic traits, namely development, also sometimes called the genotype–phenotype map. This map of genetic effects is structured in a way that enables evolvability, that is, enhances the probability that mutations will improve the performance of the organism. Here we outline two properties of organismal development, namely modularity and robustness. Modularity refers to the situation in which genes affect a restricted number of functionally related phenotypic characters. Robustness describes a situation in which cryptic mutations can accumulate without effect on fitness but can become visible to selection in a new environment or genetic background. We discuss recent empirical evidence in support of both phenomena and their effect on evolvability and also briefly address their evolution.     

Automatic detection of basal cell carcinoma by hyperspectral imaging

The purpose of this study was to test the ability of hyperspectral imaging (HSI) combined with unsupervised anomaly detectors to automatically differentiate basal cell carcinoma (BCC) from normal skin. Hyperspectral images of the face of a female patient with a BCC of the lower lip were acquired using a visible/near-infrared HSI system and two anomaly detection algorithms (Reed-Xiaoli and Reed-Xiaoli/Uniform Target hybrid anomaly detectors) were used to detect pathological tissue from normal skin. The results revealed that the receiver operating characteristic curve of the Reed-Xiaoli/Uniform Target hybrid detector was higher than that of the Reed-Xiaoli detector in the range of false positive rates between 0 and 0.8. The area under curve values were good (0.7074 and 0.8607, respectively) with Reed-Xiaoli/Uniform Target hybrid detector performing better. In conclusion, HSI combined with either of two anomaly detectors can play a promising role in the automated screening of BCC.     

Autumn migratory orientation and displacement responses of two willow warbler subspecies (Phylloscopus trochilus trochilus and P. t. acredula) in South Sweden

Topography and historical range expansion has formed a so-called migratory divide between two subspecies of willow warbler (Phylloscopus trochilus) in central Scandinavia. The autumn migratory directions of individuals assigned molecularly to both subspecies and possible hybrids were recorded using orientation cage experiments in southwest and southeast Sweden. We found pronounced differences in willow warblers’ orientation in respect to genotype. The mean directions registered in the control experiments were in accordance with the ringing recoveries and analyses of stable isotopes for Scandinavian willow warblers. With the same individuals we performed displacement experiments between both sites. They resulted in non-significant orientation, which could be explained by the intermediate distance of the displacement or reactions to housing, transportation and location. On a separate set of birds we tested whether stress following transportation could explain the disorientation and found that orientation before and after transport was unchanged. Experimental studies of effects of intermediate displacements across longitudes and studies of orientation of hybrid individuals in the zones of migratory divides are crucial for understanding the mechanisms underlying orientation behaviour. Our work further stresses the importance of knowing the migration genotype of a particular bird under study, in order to correctly evaluate expected migration routes.     

Species-specific interaction of EA1 with the maize pollen tube apex

The egg apparatus-secreted polymorphic EA1 peptide is required for micropylar pollen tube (PT) guidance in maize, the last step of the PT journey during the double fertilization process in flowering plants. In a recent study we have shown that maize PTs are attracted in vitro by EA1 and that their growth is arrested at high peptide concentrations. Moreover, we have also shown that maize PTs are guided in vitro in a species-preferential manner to the micropylar opening of transgenic Arabidopsis ovules secreting the EA1-GFP fusion protein. In support of these findings, we have improved the ligand-receptor labeling assay and report here that the EA1 peptide interacts in vitro with the maize PT apex in a species-specific manner. Bound peptide gets internalized in large vesicles and is degraded. This finding indicates that the pollen tube remains sensitive to the attractant by its rapid internalization.     

Building a synapse

L-glutamate is the primary neurotransmitter at excitatory synapses in the vertebrate CNS and at arthropod neuromuscular junctions (NMJs). However, the molecular mechanisms that trigger the recruitment of glutamate receptors at the onset of synaptogenesis and promote their stabilization at postsynaptic densities remain poorly understood. We have reported the discovery of a novel, evolutionary conserved molecule, Neto, essential for clustering of ionotropic glutamate receptors (iGluRs) at Drosophila NMJ. Neto is the first auxiliary subunit described in Drosophila and is the only non-channel subunit absolutely required for functional iGluRs. Here we review the role of Drosophila Neto in synapse assembly, its similarities with other Neto proteins and a new perspective on how glutamatergic synapses are physically assembled and stabilized.     

Polymer properties of softwood organosolv lignins produced in two different reactor systems

Lignin, the second most abundant biopolymer on earth and with a predominantly aromatic structure, has the potential to be a raw material for valuable chemicals and other bio-based chemicals. In industry, lignin is underutilized by being used mostly as a fuel for producing thermal energy. Valorization of lignin requires knowledge of the structure and different linkages in the isolated lignin, making the study of structure of lignin important. In this article, lignin samples isolated from two types of reactors (autoclave reactor and displacement reactor) were analyzed by FT-IR, size exclusion chromatography, thermogravimetric analysis (TGA), and Py-GC-MS. The average molecular mass of the organosolv lignins isolated from the autoclave reactor decreased at higher severities, and FT-IR showed an increase in free phenolic content with increasing severity. Except for molecular mass and molecular mass dispersity, there were only minor differences between lignins isolated from the autoclave reactor and lignins isolated from the displacement reactor. Carbohydrate analysis, Py-GC–MS and TGA showed that the lignin isolated using either of the reactor systems is of high purity, suggesting that organosolv lignin is a good candidate for valorization.