Interactions of an anionic antimicrobial peptide with Staphylococcus aureus membranes

The antimicrobial activity of the anionic peptide, AP1 (GEQGALAQFGEWL), was investigated. AP1 was found to kill Staphylococcus aureus with an MLC of 3 mM and to induce maximal surface pressure changes of 3.8 mN m⁻¹ over 1200 s in monolayers formed from lipid extract of S. aureus membranes. FTIR spectroscopy showed the peptide to be α-helical (100%) in the presence of vesicles formed from this lipid extract and to induce increases in their fluidity (Δν circa 0.5 cm⁻¹). These combined data show that AP1 is able to function as an α-helical antimicrobial peptide against Gram-positive bacteria and suggest that the killing mechanism used by the peptide involves interactions with the membrane lipid headgroup region. Moreover, this killing mechanism differs strongly from that previously reported for AP1 against Gram-negative bacteria, indicating the importance of considering the effects of membrane lipid composition when investigating the structure/function relationships of antimicrobial peptides.     

Nitrogen deposition enhances moss growth,but leads to an overall decline in habitat condition of mountain moss‐sedge heath

Ecosystems are subject to multiple, natural and anthropogenic environmental influences, including nitrogen (N) deposition, land use and climate. Assessment of the relative importance of these influences on biodiversity and ecosystem functioning is crucial for guiding policy and management decisions to mitigate global change; yet, few studies consider multiple drivers. In the UK, ongoing loss of the internationally important arctic/alpine moss‐sedge community, Racomitrium heath, has been linked to elevated N deposition, high grazing pressures and their combination; however, the relative importance of these drivers remains unclear. We used environmental gradients across the habitat's European distribution (UK, Faroes, Norway and Iceland) to investigate the relative impact of N deposition and grazing pressure, as well as climate, on the condition of the dominant moss species, Racomitrium lanuginosum. Key variables including tissue chemistry, growth and cover were measured at 36 sites, and multiple linear regressions were used to examine the relative importance of the drivers across sites. Our results clearly show that regional variation in the condition of R. lanuginosum across Europe is primarily associated with the impacts of N deposition, with climate (air temperature) and grazing pressure playing secondary roles. In contrast to previous experimental studies, we found moss growth to be stimulated by elevated N deposition; this apparent discrepancy may result from the use of artificially high N concentrations in many experiments. Despite increased growth rates, we found that moss mat depth and cover declined in response to N deposition. Our results suggest that this is due to increased decomposition of material in the moss mat, which ultimately leads to loss of moss cover and habitat degradation. This study clearly demonstrates both the key role of N deposition in degradation of Racomitrium heath and the importance of observational studies along natural gradients for testing predictions from experimental studies in the real world.     

Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.     

Adapting Stand‐Alone Renewable Energy Technologies for the Circular Economy through Eco‐Design and Recycling

Renewable energy (RE) technologies are looked upon favorably to provide for future energy demands and reduce greenhouse gas (GHG) emissions. However, the installation of these technologies requires large quantities of finite material resources. We apply life cycle assessment to 100 years of electricity generation from three stand‐alone RE technologies—solar photovoltaics, run‐of‐river hydro, and wind—to evaluate environmental burden profiles against baseline electricity generation from fossil fuels. We then devised scenarios to incorporate circular economy (CE) improvements targeting hotspots in systems’ life cycle, specifically (1) improved recycling rates for raw materials and (ii) the application of eco‐design. Hydro presented the lowest environmental burdens per kilowatt‐hour of electricity generation compared with other RE technologies, owing to its higher efficiency and longer life spans for main components. Distinct results were observed in the environmental performance of each system based on the consideration of improved recycling rates and eco‐design. CE measures produced similar modest savings in already low GHG emissions burdens for each technology, while eco‐design specifically had the potential to provide significant savings in abiotic resource depletion. Further research to explore the full potential of CE measures for RE technologies will curtail the resource intensity of RE technologies required to mitigate climate change.     

The Herpesvirus capsid surface protein, VP26, and the majority of the tegument proteins are dispensable for capsid transport toward the nucleus

Upon entering a cell, alphaherpesvirus capsids are transported toward the minus ends of microtubules and ultimately deposit virus DNA within the host nucleus. The virus proteins that mediate this centripetal transport are unknown but are expected to be either viral tegument proteins, which are a group of capsid-associated proteins, or a surface component of the capsid itself. Starting with derivatives of pseudorabies virus that encode a fluorescent protein fused to a structural component of the virus, we have made a collection of 12 mutant viruses that lack either the VP26 capsid protein or an individual tegument protein. Using live-cell fluorescence microscopy, we tracked individual virus particles in axons following infection of primary sensory neurons. Quantitative analysis of the VP26-null virus indicates that this protein plays no observable role in capsid transport. Furthermore, viruses lacking tegument proteins that are nonessential for virus propagation in cell culture were also competent for axonal transport. These results indicate that a protein essential for viral propagation mediates transport of the capsid to the nucleus.     

Assessing strategies for improved superfamily recognition

There are more than 200 completed genomes and over 1 million nonredundant sequences in public repositories. Although the structural data are more sparse (approximately 13,000 nonredundant structures solved to date), several powerful sequence-based methodologies now allow these structures to be mapped onto related regions in a significant proportion of genome sequences. We review a number of publicly available strategies for providing structural annotations for genome sequences, and we describe the protocol adopted to provide CATH structural annotations for completed genomes. In particular, we assess the performance of several sequence-based protocols employing Hidden Markov model (HMM) technologies for superfamily recognition, including a new approach (SAMOSA [sequence augmented models of structure alignments]) that exploits multiple structural alignments from the CATH domain structure database when building the models. Using a data set of remote homologs detected by structure comparison and manually validated in CATH, a single-seed HMM library was able to recognize 76% of the data set. Including the SAMOSA models in the HMM library showed little gain in homolog recognition, although a slight improvement in alignment quality was observed for very remote homologs. However, using an expanded 1D-HMM library, CATH-ISL increased the coverage to 86%. The single-seed HMM library has been used to annotate the protein sequences of 120 genomes from all three major kingdoms, allowing up to 70% of the genes or partial genes to be assigned to CATH superfamilies. It has also been used to recruit sequences from Swiss-Prot and TrEMBL into CATH domain superfamilies, expanding the CATH database eightfold.     

Mechanical stiffness as an improved single-cell indicator of osteoblastic human mesenchymal stem cell differentiation

Although it has been established that cellular stiffness can change as a stem cell differentiates, the precise relationship between cell mechanics and other phenotypic properties remains unclear. Inherent cell heterogeneity and asynchronous differentiation complicate population analysis; therefore, single-cell analysis was employed to determine how changes in cell stiffness correlate with changes in molecular biomarkers during differentiation. Design of a custom gridded tissue culture dish facilitated single-cell comparisons between cell mechanics and other differentiation biomarkers by enabling sequential measurement of cell mechanics and protein biomarker expression at the single cell level. The Young’s modulus of mesenchymal stem cells was shown not only to decrease during chemically-induced osteoblast differentiation, but also to correlate more closely with the day of differentiation than did the relative expression of the traditional osteoblast differentiation markers, bone sialoprotein and osteocalcin. Therefore, cell stiffness, a measurable property of individual cells, may serve as an improved indicator of single-cell osteoblast differentiation compared to traditional biological markers. Revelation of additional osteoblast differentiation indicators, such as cell stiffness, can improve identification and collection of starting cell populations, with applications to mesenchymal stem cell therapies and stem cell-based tissue engineering.     

Conservation implications for dingoes from the maternal and paternal genome: Multiple populations,dog introgression,and demography

It is increasingly common for apex predators to face a multitude of complex conservation issues. In Australia, dingoes are the mainland apex predator and play an important role in ecological functioning. Currently, however, they are threatened by hybridization with modern domestic dogs in the wild. As a consequence, we explore how increasing our understanding of the evolutionary history of dingoes can inform management and conservation decisions. Previous research on whole mitochondrial genome and nuclear data from five geographical populations showed evidence of two distinct lineages of dingo. Here, we present data from a broader survey of dingoes around Australia using both mitochondrial and Y chromosome markers and investigate the timing of demographic expansions. Biogeographic data corroborate the presence of at least two geographically subdivided genetic populations, southeastern and northwestern. Demographic modeling suggests that dingoes have undergone population expansion in the last 5,000 years. It is not clear whether this stems from expansion into vacant niches after the extinction of thylacines on the mainland or indicates the arrival date of dingoes. Male dispersal is much more common than female, evidenced by more diffuse Y haplogroup distributions. There is also evidence of likely historical male biased introgression from domestic dogs into dingoes, predominately within southeastern Australia. These findings have critical practical implications for the management and conservation of dingoes in Australia; particularly a focus must be placed upon the threatened southeastern dingo population.     

Structure and architecture of the maize genome

Maize (Zea mays or corn) plays many varied and important roles in society. It is not only an important experimental model plant, but also a major livestock feed crop and a significant source of industrial products such as sweeteners and ethanol. In this study we report the systematic analysis of contiguous sequences of the maize genome. We selected 100 random regions averaging 144 kb in size, representing about 0.6% of the genome, and generated a high-quality dataset for sequence analysis. This sampling contains 330 annotated genes, 91% of which are supported by expressed sequence tag data from maize and other cereal species. Genes averaged 4 kb in size with five exons, although the largest was over 59 kb with 31 exons. Gene density varied over a wide range from 0.5 to 10.7 genes per 100 kb and genes did not appear to cluster significantly. The total repetitive element content we observed (66%) was slightly higher than previous whole-genome estimates (58%-63%) and consisted almost exclusively of retroelements. The vast majority of genes can be aligned to at least one sequence read derived from gene-enrichment procedures, but only about 30% are fully covered. Our results indicate that much of the increase in genome size of maize relative to rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) is attributable to an increase in number of both repetitive elements and genes.