The uptake and metabolism of uridine by the slime mould Physarum polycephalum.

1. Uridine is taken up by microplasmodia of Physarum polycephalum via a saturatable transport system with an apparent Km of 29 muM. An intracellular concentration significantly higher than that in the growth medium is attained, suggesting that the uptake is an active process. Both deoxyribonucleosides and ribonucleosides are competitive inhibitors of the uptake of uridine. 2. In contrast, the rate of entry of uridine into surface plasmodia is a linear function of the concentration of the nucleoside in the growth medium, and the uptake is not inhibited by other nucleosides. 3. As well as serving as a source of pyrimidine nucleotides for the synthesis of nucleic acids, uridine is also catabolised by P. polycephalum. Uracil accumulates in the growth medium and there is also significant conversion of C-2 of the pyrimidine ring to CO2. The proportion of uridine subject to catabolism in surface plasmodia is less than that observed for microplasmodia.     

Rapid amplification and cloning of Tn5 flanking fragments by inverse PCR

A simple approach is described to efficiently amplify DNA sequences flanking transposon Tn5 insertions. The method involves: (i) digestion with a restriction enzyme that cuts within Tn5; (ii) self-ligation under conditions favouring the production of monomeric circles; (iii) four parallel PCR reactions using primers designed to amplify left or right flanking sequences, and to distinguish target amplicons from non-specific products. This reveals the number of Tn5 insertions and the size of flanking genomic restriction fragments, without Southern blot analysis. The amplified product contains restriction sites that facilitate cohesive-end cloning. This rapid method is demonstrated using Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in Xanthomonas albilineans. It is generally applicable for efficient recovery of DNA sequences flanking transposon Tn5 derivatives in insertional mutagenesis studies.     

Exposed cranial implants--a salvage operation.

We present our rationale for attempting to cover some exposed cranial implants by tissue transfers. We have tried this in 9 patients, and we have had success in 5.     

Microdiversity and temporal dynamics of marine bacterial dimethylsulfoniopropionate genes

Dimethylsulfoniopropionate (DMSP) is an abundant organic sulfur metabolite produced by many phytoplankton species and degraded by bacteria via two distinct pathways with climate-relevant implications. We assessed the diversity and abundance of bacteria possessing these pathways in the context of phytoplankton community composition over a 3-week time period spanning September–October, 2014 in Monterey Bay, CA. The dmdA gene from the DMSP demethylation pathway dominated the DMSP gene pool and was harboured mostly by members of the alphaproteobacterial SAR11 clade and secondarily by the Roseobacter group, particularly during the second half of the study. Novel members of the DMSP-degrading community emerged from dmdA sequences recovered from metagenome assemblies and single-cell sequencing, including largely uncharacterized gammaproteobacteria and alphaproteobacteria taxa. In the DMSP cleavage pathway, the SAR11 gene dddK was the most abundant early in the study, but was supplanted by dddP over time. SAR11 members, especially those harbouring genes for both DMSP degradation pathways, had a strong positive relationship with the abundance of dinoflagellates, and DMSP-degrading gammaproteobacteria co-occurred with haptophytes. This in situ study of the drivers of DMSP fate in a coastal ecosystem demonstrates for the first time correlations between specific groups of bacterial DMSP degraders and phytoplankton taxa.     

Targeting the SASP to combat ageing: Mitochondria as possible intracellular allies?

Anti‐senescence therapies, such as drugs that specifically kill senescent cells, to stave off ageing are currently under investigation. While these interventions show promise, their potential pitfalls are discussed herein. We have shown that the mitochondria are essential for development of senescence and many of the associated phenotypes, including the often detrimental senescence‐associated secretory phenotype (SASP). Here, we disentangle many ways in which the mitochondria may influence senescence and development of the SASP and focus on possible pathways that could be exploited for future generation of anti‐senescence therapies with a clear aim; to specifically eliminate the problematic features of senescent cells, while maintaining their beneficial characteristics.     

Network‐based visualisation reveals new insights into transposable element diversity

Transposable elements (TEs) are widespread across eukaryotic genomes, yet their content varies widely between different species. Factors shaping the diversity of TEs are poorly understood. Understanding the evolution of TEs is difficult because their sequences diversify rapidly and TEs are often transferred through non‐conventional means such as horizontal gene transfer. We developed a method to track TE evolution using network analysis to visualise TE sequence and TE content across different genomes. We illustrate our method by first using a monopartite network to study the sequence evolution of Tc1/mariner elements across focal species. We identify a connection between two subfamilies associated with convergent acquisition of a domain from a protein‐coding gene. Second, we use a bipartite network to study how TE content across species is shaped by epigenetic silencing mechanisms. We show that the presence of Piwi‐interacting RNAs is associated with differences in network topology after controlling for phylogenetic effects. Together, our method demonstrates how a network‐based approach can identify hitherto unknown properties of TE evolution across species.     

Generation and maintenance of diversity in the cattle MHC class I region

Major histocompatibility complex (MHC) class I genes play a crucial role in the immune defence against intracellular pathogens. An important evolutionary strategy is to generate and maintain a high level of diversity in these genes. Humans express three highly polymorphic classical MHC class I genes (HLA-A, HLA-B and HLA-C). In contrast, some species, for example rat and rhesus macaque, maintain diversity by generation of haplotypes that vary considerably with regard to the number and combination of transcribed genes. Cattle appear to use both strategies. We show that various combinations of six apparently classical genes, three of which are highly polymorphic, are transcribed on different haplotypes. Although additional sequences were identified in both cDNA and gDNA, it was not possible to assign them to any of these defined genes. Most were highly divergent or were non-classical class I genes. Thus, we found little evidence for frequent duplication and deletion of classical class I genes as reported in some other species. However, the maintenance of class I diversity in cattle may involve limited gene shuffling and deletion, possibly as a result of unequal crossing-over within the class I region.The first two authors made an equal contribution to this work.     

Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities

The folding, transport and modification of recombinant proteins in the constitutive secretory pathway of eukaryotic cell expression systems are reported to be a bottleneck in their production. We have utilised a proteomic approach to investigate the processes catalysed by proteins constituting the secretory pathway to further our understanding of those processes involved in high-level antibody secretion. We used GS-NS0 cell populations differing in qmAb to prepare enriched microsome fractions from each cell population at mid-exponential growth phase. These were analysed by 2-D PAGE to characterise the microsome protein component and test the hypothesis that bottlenecks in recombinant protein synthesis exist in these compartments, which are alleviated in high producers by the up-regulation of key secretory pathway proteins. Proteins whose abundance changed in a statistically significant manner with increasing qmAb were involved in a range of cellular functions: energy metabolism, mAb folding/assembly, cytoskeletal organisation and protein turnover. Amongst these were BiP and PDI, chaperones resident in the ER that interact with nascent immunoglobulins during their folding/assembly. However, our results suggest that there are diverse mechanisms by which these cells achieve qmAb. The results imply that cell-engineering strategies for improving qmAb should target proteins associated with altered functional phenotype identified in this study.     

A Comparison of Soil Microbial Community Structure, Protozoa and Nematodes in Field Plots of Conventional and Genetically Modified Maize Expressing the Bacillus thuringiens is CryIAb Toxin

Field trials were established at three European sites (Denmark, Eastern France, South-West France) of genetically modified maize (Zea mays L.) expressing the CryIAb Bacillus thuringiensis toxin (Bt), the near-isogenic non-Bt cultivar, another conventional maize cultivar and grass. Soil from Denmark was sampled at sowing (May) and harvest (October) over two years (2002, 2003); from E France at harvest 2002, sowing and harvest 2003; and from SW France at sowing and harvest 2003. Samples were analysed for microbial community structure (2003 samples only) by community-level physiological-profiling (CLPP) and phospholipid fatty acid analysis (PLFA), and protozoa and nematodes in all samples. Individual differences within a site resulted from: greater nematode numbers under grass than maize on three occasions; different nematode populations under the conventional maize cultivars once; and two occasions when there was a reduced protozoan population under Bt maize compared to non-Bt maize. Microbial community structure within the sites only varied with grass compared to maize, with one occurrence of CLPP varying between maize cultivars (Bt versus a conventional cultivar). An overall comparison of Bt versus non-Bt maize across all three sites only revealed differences for nematodes, with a smaller population under the Bt maize. Nematode community structure was different at each site and the Bt effect was not confined to specific nematode taxa. The effect of the Bt maize was small and within the normal variation expected in these agricultural systems.