Overcoming barriers to membrane protein structure determination

After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.     

Overcoming barriers to understanding the cellular basis of aluminium resistance

There appears to be an emerging consensus that resistance to aluminium (Al) is mediated at the cellular level. Virtually all current hypotheses which seek to explain the basis of Al resistance have a cellular focus, including those which postulate that external mechanisms limit the rate of Al entry across the membrane and/or protect sensitive extracellular sites, as well as those which postulate that internal mechanisms detoxify Al in the cytoplasm. If Al resistance is a cellular phenomenon, it should be expressed in single cells. Attempts to demonstrate resistance in cell culture systems, however, have not been uniformly satisfying. Considerable uncertainty has arisen from use of experimental conditions which favour formation of insoluble or non-toxic Al species. This problem has plagued research which has attempted to select for Al resistance in cell culture systems, as well as research which has attempted to express existing patterns of differential resistance in cell culture systems. Despite technical problems such as this, work at the cellular level has provided some important contributions. Most importantly, we now know resistance to Al can be expressed at the cellular level. We have discovered also that plant cells accumulate Al much more rapidly in cell culture systems than in intact roots and that isolated cells are more sensitive to Al than complex tissues. While this type of research is still hampered by a number of technical barriers, it would appear that more rapid progress could be achieved if greater emphasis was placed on true experimental work. Furthermore, we need to begin evaluating experimental data in the context of an integrated Al stress response if we are to achieve a full understanding of the cellular basis of Al resistance.     

Overcoming protein diversity on arrays

Researchers show that to reliably establish protein interaction networks from microarray data it is necessary to measure saturation binding curves for each arrayed protein and its potential binding partners.     

Overcoming natural replication barriers: differential helicase requirements

DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.     

Overcoming physiologic barriers to cancer treatment by molecularly targeting the tumor microenvironment

It is widely recognized that the vasculature of the tumor is inadequate to meet the demands of the growing mass. The malformed vasculature is at least in part responsible for regions of the tumor that are hypoxic, acidotic, and exposed to increased interstitial fluid pressure. These unique aspects of the tumor microenvironment have been shown to act as barriers to conventional chemotherapy or radiation-based therapies. It now seems that while the vasculature initiates these tumor-specific conditions, the cells within the tumor respond to these stresses and add to the unique solid tumor physiology. Gene expression changes have been reported in the tumor for vascular endothelial growth factor, carbonic anhydrase IX, and pyruvate dehydrogenase kinase 1. The activity of these gene products then influences the tumor physiology through alterations in vascular permeability and interstitial fluid pressure, extracellular acidosis, and mitochondrial oxygen consumption and hypoxia, respectively. Novel molecular strategies designed to interfere with the activities of these gene products are being devised as ways to overcome the physiologic barriers in the tumor to standard anticancer therapies.     

A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems

BACKGROUND: Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the diversity of total cells and active cells. METHODS: Total bacteria were determined by SYBR-II staining. Viable bacteria were determined in water samples from different sites by plate count techniques and by the direct viable count (DVC) method. Substrate-responsive cells (i.e., DVC(+) cells) were distinguished from nonresponsive cells (i.e., DVC(-) cells) by FCM and sorted. The genetic diversity of the sorted cell fraction was compared with the diversity of the total microbial community and with that of the culturable cell fraction by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. The same approach was applied to a seawater sample enriched with nutrients. In this case, actively respiring cells (CTC+) were also enumerated by FCM, sorted, and analyzed by DGGE. RESULTS: The diversity of viable cells varied depending on the methods (traditional culture or DVC) used for viability assessment. Some phylotypes detected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring cells. Inversely, some phylotypes found at the community level were not found in viable and active cell-sorted fractions. It suggests that diversity determined at the community level includes nonactive and nonviable cells. CONCLUSION: This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbial ecology such as the identification of species able to maintain cellular activity under environmental changes or in the presence of toxic compounds are now possible.     

Overcoming the barriers to the retirement of Old and New World monkeys from research facilities

In this commentary I aim to raise awareness among researchers and sanctuary directors to potential barriers to retiring Old and New World monkeys from research facilities. I define a barrier as an opinion or stereotype that prevents primate retirement from occurring on a regular basis. By discussing retirement barriers and recommending how to overcome them, I aim to increase the frequency of retiring monkeys from the laboratory into naturalistic sanctuaries. In this article I compile a final list of 10 barriers to retirement—and recommendations on how to overcome them—based on responses to forums, comments from primate sanctuary directors, information contained in scientific and sanctuary literature, and personal experiences. I conclude that researchers will increase the frequency of primate retirement by performing the following 5 actions: (a) increase communication by networking with sanctuaries, (b) prevent negative publicity by developing a confidentiality clause with the sanctuary, (c) increase understanding by reviewing the articles written on retiring monkeys into sanctuaries, (d) increase funding for primate retirement by including funding requests in grant proposals, or (e) by raising private funds.     

Overcoming barriers to the broader implementation of life cycle thinking in business and public administration

Preamble. It is the Purpose of this Corner to serve as an information and communication channel as well as a discussion forum between science and policy in general, as well as between research and management, in business and public administration with regard to goods and services (products). It is the aim of this Corner to improve awareness of, and to help more effectively implement, life cycle thinking in all stages of decision making in business and public administration. This Corner ‘EU Life Cycle Policy and Support’ appears regularly. Contributions are invited and uninvited articles, as well as discussion comments from the LCA community. They outline practical experiences, information on new regulations, directives and other policy instruments, as well as important theoretical considerations. This first edition of the Corner provides, for discussion, a broad commentary on our underlying thoughts and ideas. This builds on ideas presented in e.g. the business context in the Life Cycle Management special edition (Int J LCA Special Issue 1, 2007), as well as recent policy developments in the European Union. This editorial is illustrated with example references related to each issue that were recently published in the journal (see [1–11]). Dr. David W. Pennington is responsible for the Environmental Assessment of Waste and the Sustainable Management of Natural Resources (ENSURE) in the European Commission’s Joint Research Centre (JRC), based at the Institute for Environment and Sustainability in Ispra, Italy. ENSURE complements conventional methods for waste and resource management with the development of recommended approaches, indicators, reference data, and case studies that facilitate life cycle thinking in European business and public administrations. The ENSURE team, consisting of about 15 staffs, provides independent support through computer simulations, modelling, expert workshops, and state-of-the-art laboratories. The responsibilities include the implementation of the European Platform on LCA, which is developing the Reference European Life Cycle Data System (ELCD) and associated Technical Guidance Documents. The team has successfully conducted a series of international workshops and carried out pilot studies with Member States in relation to Life Cycle Thinking and Waste Management. Activities now include the development of European Life-Cycle Guidelines for Waste Management, and the broader development of Life Cycle-based Sustainability Indicators.     

Implications of recurrent disturbance for genetic diversity

Exploring interactions between ecological disturbance, species’ abundances and community composition provides critical insights for ecological dynamics. While disturbance is also potentially an important driver of landscape genetic patterns, the mechanisms by which these patterns may arise by selective and neutral processes are not well‐understood. We used simulation to evaluate the relative importance of disturbance regime components, and their interaction with demographic and dispersal processes, on the distribution of genetic diversity across landscapes. We investigated genetic impacts of variation in key components of disturbance regimes and spatial patterns that are likely to respond to climate change and land management, including disturbance size, frequency, and severity. The influence of disturbance was mediated by dispersal distance and, to a limited extent, by birth rate. Nevertheless, all three disturbance regime components strongly influenced spatial and temporal patterns of genetic diversity within subpopulations, and were associated with changes in genetic structure. Furthermore, disturbance‐induced changes in temporal population dynamics and the spatial distribution of populations across the landscape resulted in disrupted isolation by distance patterns among populations. Our results show that forecast changes in disturbance regimes have the potential to cause major changes to the distribution of genetic diversity within and among populations. We highlight likely scenarios under which future changes to disturbance size, severity, or frequency will have the strongest impacts on population genetic patterns. In addition, our results have implications for the inference of biological processes from genetic data, because the effects of dispersal on genetic patterns were strongly mediated by disturbance regimes.     

Occurrence and genetic diversity of Bacillus anthracis strains isolated in an active wool-cleaning factory

Culturable microorganisms from various samples taken at an active factory performing wool and goat hair cleaning were isolated and analyzed. Bacillus anthracis was found in air filter dust, wastewater, and goat hairs, where it accounted for approximately 1% of the total counts of viable bacteria. Consistent with the countries of origin of the processed material (South Caucasian and Middle Eastern), all B. anthracis isolates belonged to the same phylogenetic cluster, as determined by variable-number tandem repeat (VNTR) typing at eight loci. Within this cluster, five closely related VNTR subtypes could be identified, of which two were previously unreported. Additional diversity was observed when more sensitive genetic markers were assayed, demonstrating the multifocal nature of goat hair contamination. Goat hair originating from areas where anthrax is endemic remains a material with high biological risk for modern woolworkers.     

Selecting subsets of genotyped experimental populations for phenotyping to maximize genetic diversity

Selective phenotyping is a way of capturing the benefits of large population sizes without the need to carry out large-scale phenotyping and hence is a cost-effective means of capturing information about gene–trait relationships within a population. The diversity within the sample gives an indication of the efficiency of this information capture; less diversity implies greater redundancy of the genetic information. Here, we propose a method to maximize genetic diversity within the selected samples. Our method is applicable to general experimental designs and robust to common problems such as missing data and dominant markers. In particular, we discuss its application to multi-parent advanced generation intercross (MAGIC) populations, where, although thousands of lines may be genotyped as a large population resource, only hundreds may need to be phenotyped for individual studies. Through simulation, we compare our method to simple random sampling and the minimum moment aberration method. While the gain in power over simple random sampling for all tested methods is not large, our method results in a much more diverse sample of genotypes. This diversity can be applied to improve fine mapping resolution once a QTL region has been detected. Further, when applied to two wheat datasets from doubled haploid and MAGIC progeny, our method detects known QTL for small sample sizes where other methods fail.     

Overcoming crossing barriers between nontuber-bearing and tuber-bearing Solanum species: towards potato germplasm enhancement with a broad spectrum of solanaceous genetic resources.

The first direct sexual hybrids between diploid nontuber-bearing species and diploid potato breeding lines are reported here. Three nontuberous species of Solanum, S. brevidens, S. etuberosum, and S. fernandezianum, were used for sexual crosses, achieved by a combination of rescue pollinations and embryo rescue. Initial hybrid selection was made using an embryo spot marker, followed by the evaluation of morphological and reproductive traits. Putative hybrids were first tested for resistance to potato leaf roll virus derived from the wild species, and then were tested with molecular markers using species-specific DNA probes. Finally, the tuberization of several 2x hybrids was tested for actual potato germplasm enhancement. These hybrids are unique in terms of their potential to enhance recombination between chromosomes of wild species and those of cultivated potatoes in germplasm utilization, and to exploit the genetic nature of tuber formation. The finding that nontuber-bearing Solanum spp. can be directly crossed with tuber-bearing species also has important implications for the regulatory aspects of the use of genetically modified organisms.     

Ecology: linking species diversity and genetic diversity

Although there is a great deal of interest in the biological diversity of species and of genes, it is only recently that researchers have begun to investigate the processes that exert parallel influences on these different levels of diversity.     

Effects of geographic distance,sea barriers and habitat on the genetic structure and diversity of all-hybrid water frog populations

The history of population size and migration patterns leaves its mark in the genetics of populations. We investigate the genetic structure of the edible frog, Pelophylax esculentus in the Danish archipelago and adjacent countries. This frog is of particular interest because it is a hybrid that, in this area, forms all-hybrid populations of diploid (LR) and triploid (LLR and LRR) genomotypes with no (or very few) adults of the parental species (LL and RR). This study is the first to cover the entire geographic range of Danish, Swedish and German all-hybrid populations, documenting their extent and providing a broad picture of their diversity of neutral genetic markers and genomotype proportions. With 18 microsatellite markers, we found that genetic diversity declines northwards in agreement with the glacial refuge and central-marginal hypotheses; however, populations on small and medium-sized islands are no less diverse than those on large islands and continental peninsulas. Isolation by distance exists across the archipelago with limited influence of fragmentation by brackish seawater. The extremely low genetic diversity in all-hybrid populations, compared with adjacent populations, may be responsible for the maintenance of their special breeding system. We also show large variation among ponds in proportions of LLR, LR and LRR genomotypes, but little geographic pattern in their distribution. Instead, we found relationships between the genomotype proportions and some of 15 habitat parameters monitored. Body size differences among LLR, LR and LRR further suggest ecological differences.     

smogd: software for the measurement of genetic diversity

Software for the measurement of genetic diversity (SMOGD) is a web-based application for the calculation of the recently proposed genetic diversity indices G'(ST) and D(est) . SMOGD includes bootstrapping functionality for estimating the variance, standard error and confidence intervals of estimated parameters, and SMOGD also generates genetic distance matrices from pairwise comparisons between populations. SMOGD accepts standard, multilocus Genepop and Arlequin formatted input files and produces HTML and tab-delimited output. This allows easy data submission, quick visualization, and rapid import of results into spreadsheet or database programs.