Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

Can behavioral and personality traits influence the success of unintentional species introductions?

Unintentional species invasions are instigated by human-mediated dispersal of individuals beyond their native range. Although most introductions fail at the first hurdle, a select subset pass through each stage of the introduction process (i.e., transport, introduction, establishment and spread) to become successful invaders. Efforts to identify the traits associated with invasion success have predominately focused on deliberate introductions, which essentially bypass the initial introduction stage. Here, we highlight how behavior influences the success or failure of unintentional species introductions across each stage of the introduction process, with a particular focus on transportation and initial establishment. In addition, we emphasize how recent advances in understanding of animal personalities and individual-level behavioral variation can help elucidate the mechanisms underlying the success of stowaways.     

Designed for deconstruction--poplar trees altered in cell wall lignification improve the efficacy of bioethanol production

? There is a pressing global need to reduce the increasing societal reliance on petroleum and to develop a bio-based economy. At the forefront is the need to establish a sustainable, renewable, alternative energy sector. This includes liquid transportation fuel derived from lignocellulosic plant materials. However, one of the current limiting factors restricting the effective and efficient conversion of lignocellulosic residues is the recalcitrance of the substrate to enzymatic conversion. ? In an attempt to assess the impact of cell wall lignin on recalcitrance, we subjected poplar trees engineered with altered lignin content and composition to two potential industrial pretreatment regimes, and evaluated the overall efficacy of the bioconversion to ethanol process. ? It was apparent that total lignin content has a greater impact than monomer ratio (syringyl : guaiacyl) on both pretreatments. More importantly, low lignin plants showed as much as a 15% improvement in the efficiency of conversion, with near complete hydrolysis of the cellulosic polymer. ? Using genomic tools to breed or select for modifications in key cell wall chemical and/or ultrastructural traits can have a profound effect on bioenergy processing. These techniques may therefore offer means to overcome the current obstacles that underpin the recalcitrance of lignocellulosic substrates to bioconversion.     

Using stable isotope analysis to understand the migration and trophic ecology of northeastern Pacific white sharks (Carcharodon carcharias)

The white shark (Carcharodon carcharias) is a wide-ranging apex predator in the northeastern Pacific (NEP). Electronic tagging has demonstrated that white sharks exhibit a regular migratory pattern, occurring at coastal sites during the late summer, autumn and early winter and moving offshore to oceanic habitats during the remainder of the year, although the purpose of these migrations remains unclear. The purpose of this study was to use stable isotope analysis (SIA) to provide insight into the trophic ecology and migratory behaviors of white sharks in the NEP. Between 2006 and 2009, 53 white sharks were biopsied in central California to obtain dermal and muscle tissues, which were analyzed for stable isotope values of carbon (δ(13)C) and nitrogen (δ(15)N). We developed a mixing model that directly incorporates movement data and tissue incorporation (turnover) rates to better estimate the relative importance of different focal areas to white shark diet and elucidate their migratory behavior. Mixing model results for muscle showed a relatively equal dietary contribution from coastal and offshore regions, indicating that white sharks forage in both areas. However, model results indicated that sharks foraged at a higher relative rate in coastal habitats. There was a negative relationship between shark length and muscle δ(13)C and δ(15)N values, which may indicate ontogenetic changes in habitat use related to onset of maturity. The isotopic composition of dermal tissue was consistent with a more rapid incorporation rate than muscle and may represent more recent foraging. Low offshore consumption rates suggest that it is unlikely that foraging is the primary purpose of the offshore migrations. These results demonstrate how SIA can provide insight into the trophic ecology and migratory behavior of marine predators, especially when coupled with electronic tagging data.     

Relaxation of selective constraints causes independent selenoprotein extinction in insect genomes

Background

Selenoproteins are a diverse family of proteins notable for the presence of the 21st amino acid, selenocysteine. Until very recently, all metazoan genomes investigated encoded selenoproteins, and these proteins had therefore been believed to be essential for animal life. Challenging this assumption, recent comparative analyses of insect genomes have revealed that some insect genomes appear to have lost selenoprotein genes.

Methodology/Principal Findings

In this paper we investigate in detail the fate of selenoproteins, and that of selenoprotein factors, in all available arthropod genomes. We use a variety of in silico comparative genomics approaches to look for known selenoprotein genes and factors involved in selenoprotein biosynthesis. We have found that five insect species have completely lost the ability to encode selenoproteins and that selenoprotein loss in these species, although so far confined to the Endopterygota infraclass, cannot be attributed to a single evolutionary event, but rather to multiple, independent events. Loss of selenoproteins and selenoprotein factors is usually coupled to the deletion of the entire no-longer functional genomic region, rather than to sequence degradation and consequent pseudogenisation. Such dynamics of gene extinction are consistent with the high rate of genome rearrangements observed in Drosophila. We have also found that, while many selenoprotein factors are concomitantly lost with the selenoproteins, others are present and conserved in all investigated genomes, irrespective of whether they code for selenoproteins or not, suggesting that they are involved in additional, non-selenoprotein related functions.

Conclusions/Significance

Selenoproteins have been independently lost in several insect species, possibly as a consequence of the relaxation in insects of the selective constraints acting across metazoans to maintain selenoproteins. The dispensability of selenoproteins in insects may be related to the fundamental differences in antioxidant defense between these animals and other metazoans.     

Genetic divergences pre-date Pleistocene glacial cycles in the New Zealand speckled skink, Oligosoma infrapunctatum

Aim To examine the hypothesis raised by Graham S. Hardy that Pleistocene glacial cycles suffice to explain divergence among lineages within the endemic New Zealand speckled skink, Oligosoma infrapunctatum Boulenger. Location Populations were sampled from across the entire range of the species, on the North and South Islands of New Zealand. Methods We sequenced the mitochondrial genes ND2 (550 bp), ND4 + tRNAs (773 bp) and cytochrome b (610 bp) of 45 individuals from 21 locations. Maximum likelihood, maximum parsimony and Bayesian methods were used for phylogenetic reconstruction. The Shimodaira–Hasegawa test was used to examine hypotheses about the taxonomic status of morphologically distinctive populations. Results Our analysis revealed four strongly supported clades within O. infrapunctatum. Clades were largely allopatric, except on the west coast of the South Island, where representatives from all four clades were found. Divergences among lineages within the species were extremely deep, reaching over 5%. Two contrasting phylogeographical patterns are evident within O. infrapunctatum. Main conclusions The deep genetic divisions we found suggest that O. infrapunctatum is a complex of cryptic species which diverged in the Pliocene, contrary to the existing Pleistocene‐based hypothesis. Although Pleistocene glacial cycles do not underlie major divergences within this species, they may be responsible for the shallower phylogeographical patterns that are found within O. infrapunctatum, which include a radiation of haplotypes in the Nelson and Westland regions.     

Improvement of biomass through lignin modification

Lignin, a major component of the cell wall of vascular plants, has long been recognized for its negative impact on forage quality, paper manufacturing, and, more recently, cellulosic biofuel production. Over the last two decades, genetic and biochemical analyses of brown midrib mutants of maize, sorghum and related grasses have advanced our understanding of the relationship between lignification and forage digestibility. This work has also inspired genetic engineering efforts aimed at generating crops with altered lignin, with the expectation that these strategies would enhance forage digestibility and/or pulping efficiency. The knowledge gained from these bioengineering efforts has greatly improved our understanding of the optimal lignin characteristics required for various applications of lignocellulosic materials while also contributing to our understanding of the lignin biosynthetic pathway. The recent upswing of interest in cellulosic biofuel production has become the new focus of lignin engineering. Populus trichocarpa and Brachypodium distachyon are emerging as model systems for energy crops. Lignin research on these systems, as well as on a variety of proposed energy crop species, is expected to shed new light on lignin biosynthesis and its regulation in energy crops, and lead to rational genetic engineering approaches to modify lignin for improved biofuel production.     

Stigma,shame, and blame experienced by patients with lung cancer: qualitative study

Objectives To draw on narrative interviews with patients with lung cancer and to explore their perceptions and experience of stigma.Design Qualitative study.Setting United Kingdom.Participants 45 patients with lung cancer recruited through several sources.Results Participants experienced stigma commonly felt by patients with other types of cancer, but, whether they smoked or not, they felt particularly stigmatised because the disease is so strongly associated with smoking. Interaction with family, friends, and doctors was often affected as a result, and many patients, particularly those who had stopped smoking years ago or had never smoked, felt unjustly blamed for their illness. Those who resisted victim blaming maintained that the real culprits were tobacco companies with unscrupulous policies. Some patients concealed their illness, which sometimes had adverse financial consequences or made it hard for them to gain support from other people. Some indicated that newspaper and television reports may have added to the stigma: television advertisements aim to put young people off tobacco, but they usually portray a dreadful death, which may exacerbate fear and anxiety. A few patients were worried that diagnosis, access to care, and research into lung cancer might be adversely affected by the stigma attached to the disease and those who smoke.Conclusion Patients with lung cancer report stigmatisation with far reaching consequences. Efforts to help people to quit smoking are important, but clinical and educational interventions should be presented with care so as not to add to the stigma experienced by patients with lung cancer and other smoking related diseases.