Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost

Automation and miniaturization are key issues of high-throughput research projects in the post-genomic era. The implementation of robotics and parallelization has enabled researchers to process large numbers of protein targets for structural studies in a short time with reasonable cost efficiency. However, the cost of implementing the robotics and parallelization often prohibit their use in the traditional academic laboratory. Fortunately, multiple groups have made significant efforts to minimize the cost of heterologous protein expression for the production of protein samples in quantities suitable for high resolution structural studies. In this review, we describe recent efforts to continue to minimize the cost for the parallel processing of multiple protein targets and focus on those materials and strategies that are highly suitable for the traditional academic laboratory.     

The utility of low-density genotyping for imputation in the Thoroughbred horse

Background

Despite the dramatic reduction in the cost of high-density genotyping that has occurred over the last decade, it remains one of the limiting factors for obtaining the large datasets required for genomic studies of disease in the horse. In this study, we investigated the potential for low-density genotyping and subsequent imputation to address this problem.

Results

Using the haplotype phasing and imputation program, BEAGLE, it is possible to impute genotypes from low- to high-density (50K) in the Thoroughbred horse with reasonable to high accuracy. Analysis of the sources of variation in imputation accuracy revealed dependence both on the minor allele frequency of the single nucleotide polymorphisms (SNPs) being imputed and on the underlying linkage disequilibrium structure. Whereas equidistant spacing of the SNPs on the low-density panel worked well, optimising SNP selection to increase their minor allele frequency was advantageous, even when the panel was subsequently used in a population of different geographical origin. Replacing base pair position with linkage disequilibrium map distance reduced the variation in imputation accuracy across SNPs. Whereas a 1K SNP panel was generally sufficient to ensure that more than 80% of genotypes were correctly imputed, other studies suggest that a 2K to 3K panel is more efficient to minimize the subsequent loss of accuracy in genomic prediction analyses. The relationship between accuracy and genotyping costs for the different low-density panels, suggests that a 2K SNP panel would represent good value for money.

Conclusions

Low-density genotyping with a 2K SNP panel followed by imputation provides a compromise between cost and accuracy that could promote more widespread genotyping, and hence the use of genomic information in horses. In addition to offering a low cost alternative to high-density genotyping, imputation provides a means to combine datasets from different genotyping platforms, which is becoming necessary since researchers are starting to use the recently developed equine 70K SNP chip. However, more work is needed to evaluate the impact of between-breed differences on imputation accuracy.     

Prey selection by coral-feeding butterflyfishes: strategies to maximize the profit

Synopsis Factors that structure preferences among food corals were examined for the obligate coral-feeding butterflyfishChaetodon multicinctus. In the field, fish show a simple repetitious pattern of foraging composed of (1) pre-encounter search for coral colonies, and (2) post-encounter inspection/orientation, bite, and consumption of polyps. Rose coral,Pocillopora meandrina, and the massive coral,Porites lobata, were taken in higher proportions than their percentage substrate cover, while finger coral,Porites compressa, was taken in lower proportion. Paired presentations of coral colonies in the lab gave similar results:Poc. meandrina was preferred overPor. lobata which was preferred overPor. compressa. Poc. meandrina tissue had the highest energy content, lowest handling time, and highest profitability. Energy content did not differ amongPorites tissues, but handling time was greater and more inspective eye movements were made while foraging on the branched finger coral,Por. compressa. Experimental manipulation of coral colony morphology indicate preferences amongPorites are most likely structured by handling costs. Predictions of a simple prey-choice foraging model are supported in theC. multicinctus system if abundance of the branched coralPor. compressa is estimated as that available to fishes rather than percentage substrate cover. The relative size and abundance of stinging nematocysts are also consistent with observed foraging patterns in the field, but await immunological confirmation. Coral-feeding butterflyfishes offer unique opportunities to test models of foraging ecology in reef fishes, and the direction of future studies is suggested.     

Analyses and comparison of accuracy of different genotype imputation methods

The power of genetic association analyses is often compromised by missing genotypic data which contributes to lack of significant findings, e.g., in in silico replication studies. One solution is to impute untyped SNPs from typed flanking markers, based on known linkage disequilibrium (LD) relationships. Several imputation methods are available and their usefulness in association studies has been demonstrated, but factors affecting their relative performance in accuracy have not been systematically investigated. Therefore, we investigated and compared the performance of five popular genotype imputation methods, MACH, IMPUTE, fastPHASE, PLINK and Beagle, to assess and compare the effects of factors that affect imputation accuracy rates (ARs). Our results showed that a stronger LD and a lower MAF for an untyped marker produced better ARs for all the five methods. We also observed that a greater number of haplotypes in the reference sample resulted in higher ARs for MACH, IMPUTE, PLINK and Beagle, but had little influence on the ARs for fastPHASE. In general, MACH and IMPUTE produced similar results and these two methods consistently outperformed fastPHASE, PLINK and Beagle. Our study is helpful in guiding application of imputation methods in association analyses when genotype data are missing.     

Evaluation of alternative strategies to optimize ketorolac transdermal delivery

In the present study, 2 alternative strategies to optimize ketorolac transdermal delivery, namely, prodrugs (polyoxyethylene glycol ester derivatives, I–IV) and nanostructured lipid carriers (NLC) were investigated. The synthesized prodrugs were chemically stable and easily degraded to the parent drug in human plasma. Ketorolac-loaded NLC with high drug content could be successfully prepared. The obtained products formulated into gels showed a different trend of drug permeation through human stratum corneum and epidermis. Particularly, skin permeation of ester prodrugs was significantly enhanced, apart from ester IV, compared with ketorolac, while the results of drug release from NLC outlined that these carriers were ineffective in increasing ketorolac percutaneous absorption owing to a higher degree of mutual interaction between the drug and carrier lipid matrix. Polyoxyethylene glycol esterification confirmed to be a suitable approach to enhance ketorolac transdermal delivery, while NLC seemed more appropriate for sustained release owing to the possible formation of a drug reservoir into the skin. Published: August 4, 2006     

An alternative genotyping method using dye-labeled universal primer to reduce unspecific amplifications

We proposed a modification the procedure of genotyping based in labeled universal primer and tailed primer. In the standard protocol, three primers are used in the same PCR reaction, a forward primer with tail added at the 5′ end of the identical sequence to labeled universal primer with dye-fluorescent and a reverse primer. Unfortunately, the choice of a labeled primer characterized by a large number of complementary sequences in target genomes (which is more probable in larger genomes) result in unspecific amplifications (false positive) can cause absence or decrease amplification of the locus of interest and also false interpretation of the analysis. However, identification of possible homologies between the primer chosen for labelling and the genome is rarely possible from the available DNA data bases. In our approach, cycling is interrupted for the addition of the labeled primer only during the final cycles, thus minimizing unspecific amplification and competition between primers, resulting in the more fidelity amplification of the target regions.     

A comparison between SNaPshot, pyrosequencing, and biplex invader SNP genotyping methods: accuracy, cost, and throughput

Three methods of Single Nucleotide Polymorphism (SNP) detection: SNaPshot, Pyrosequencing and Biplex Invader, with two different chemistries were investigated to compare, (1) accuracy, (2) ease of use, (3) throughput capability, and (4) cost. We genotyped 192 human DNA samples across 24 SNPs (minor allele frequencies above 30%), of which seven SNPs were genotyped with all three methods. We show that the Biplex Invader genotyping method was found to be the most accurate and easiest to use with lowest cost, although Pyrosequencing provided similar results at a low cost. With little optimization, the accuracy of the SNaPshot method was also comparable to these two methods with a higher cost, if only singleplex reactions are used.     

A comparison of strategies for selecting breeding pairs to maximize genetic diversity retention in managed populations

Captive breeding programs aim to maintain populations that are demographically self-sustaining and genetically healthy. It has been well documented that the best way for managed breeding programs to retain gene diversity (GD) and limit inbreeding is to select breeding pairs that minimize a population's average kinship. We used a series of computer simulations to test 4 methods of minimizing average kinship across a variety of scenarios with varying generation lengths, mortality rates, reproductive rates, and rates of breeding pair success. "Static MK Selection" and "Dynamic MK Selection" are 2 methods for iteratively selecting genetically underrepresented individuals for breeding, whereas "Ranked MK Selection" and "Simultaneous MK Selection" are 2 methods for concurrently selecting the group of breeding individuals that produce offspring with the lowest average kinship. For populations with discrete generations (24 tested scenarios), we found that the Simultaneous and Ranked MK Selection methods were generally the best, nearly equivalent methods for selecting breeding pairs that retained GD and limited inbreeding. For populations with overlapping generations (198 tested scenarios), we found that Dynamic MK Selection was the most robust method for selecting breeding pairs. We used these results to provide guidelines for identifying which method of minimizing average kinship was most appropriate for various breeding program scenarios.     

How initiation factors maximize the accuracy of tRNA selection in initiation of bacterial protein synthesis

During initiation of bacterial protein synthesis, messenger RNA and fMet-tRNAfMet bind to the 30S ribosomal subunit together with initiation factors IF1, IF2, and IF3. Docking of the 30S preinitiation complex to the 50S ribosomal subunit results in a peptidyl-transfer competent 70S ribosome. Initiation with an elongator tRNA may lead to frameshift and an aberrant N-terminal sequence in the nascent protein. We show how the occurrence of initiation errors is minimized by (1) recognition of the formyl group by the synergistic action of IF2 and IF1, (2) uniform destabilization of the binding of all tRNAs to the 30S subunit by IF3, and (3) an optimal distance between the Shine-Dalgarno sequence and the initiator codon. We suggest why IF1 is essential for E. coli, discuss the role of the G-C base pairs in the anticodon stem of some tRNAs, and clarify gene expression changes with varying IF3 concentration in the living cell.     

Simulated long-term vegetation response to alternative stocking strategies in savanna rangelands

Increasing cover by woody vegetation, prevalent in semiarid savanna rangelands throughout the world, is a degrading process attributed to the grazing impact as a major causal factor. We studied grazing effects on savanna vegetation dynamics under alternative stocking strategies with a spatially explicit grid-based simulation model grounded in Kalahari (southern Africa) ecology. Plant life histories were modeled for the three major life forms: perennial grasses, shrubs, annuals. We conducted simulation experiments over a range of livestock utilization intensities for three alternative scenarios of small scale grazing heterogeneity, and two alternative strategies: fixed stocking versus adaptive stocking tracking herbage production. Additionally, the impact of the duration of the management planning horizon was studied, by comparing community response and mean stocking rates after 20 and 50 years. Results confirmed a threshold behavior of shrub cover increase: at low, subcritical utilization intensity little change occurred; when utilization intensity exceeded a threshold, shrub cover increased drastically. For both stocking strategies, thresholds were highly sensitive to grazing heterogeneity. At a given critical utilization intensity, the long term effect of grazing depended on the level of grazing heterogeneity: whereas under low heterogeneity, shrub cover remained unchanged, a large increase occurred under highly heterogeneous grazing. Hence, information on spatial grazing heterogeneity is crucial for correct assessment of the impact of livestock grazing on vegetation dynamics, and thus for the assessment of management strategies. Except for the least heterogeneous grazing scenario, adaptive stocking allowed a more intensive utilization of the range without inflating the risk of shrub cover increase. A destabilizing feedback between rainfall and herbage utilization was identified as the major cause for the worse performance of fixed compared to adaptive stocking, which lacks this feedback. Given the usually high grazing heterogeneity in semiarid rangelands, adaptive stocking provides a management option for increasing herbage utilization and thus returns of livestock produce without increasing degradation risks.     

Application of organization-activation theory to alternative male reproductive strategies: a review

Many species have extreme within-sex morphological and behavioral polymorphisms, most commonly different male phenotypes that practice different reproductive strategies. Although much is known about the role of hormones in sexual differentiation, little is known about what role hormones might play in within-sex differentiation. The relative plasticity hypothesis is derived from the classical organization-activation model of hormone action. It distinguishes between two types of polymorphic systems: a fixed system in which individual males assume one phenotype for their adult lives and a plastic system in which individual males can change phenotypes at least once. By analogy to sexual differentiation, the relative plasticity hypothesis generally predicts that organizational influences of hormones will be more important in fixed systems and activational influences of hormones will be more important in plastic systems. A review of our knowledge of the role of hormones in differentiation of within-sex polymorphisms indicates that the relative plasticity hypothesis accounts for otherwise diverse and contradictory results. This further supports the hypothesis that the organizational-activational model of hormone action derived from sexual differentiation generalizes to within-sex polymorphisms. However, studies of the effects of hormone manipulations on within-sex differentiation are rare but are desperately needed to further our understanding of this problem. Further studies of discontinuous behavioral variation characteristic of polymorphic species may further our understanding of the physiological basis of within-sex behavior variation in all species.     

Assessment of methods to determine minimal cellulase concentrations for efficient hydrolysis of cellulose

Summary The enzyme loading needed to achieve substrate saturation appeared to be the most economical enzyme concentration to use for hydrolysis, based on percentage hydrolysis. Saturation was reached at 25 filter paper units per gram substrate on Solka Floc BW300, as determined by studying (a) initial adsorption of the cellulase preparation onto the substrate, (b) an actual hydrolysis or (c) a combined hydrolysis and fermentation (CHF) process. Initial adsorption of the cellulases onto the substrate can be used to determine the minimal cellulase requirements for efficient hydrolysis since enzymes initially adsorbed to the substrate have a strong role in governing the overall reaction. Trichoderma harzianum E58 produces high levels of -glucosidase and is able to cause high conversion of Solka Floc BW300 to glucose without the need for exogenous -glucosidase. End-product inhibition of the cellulase and -glucosidase can be more effectively reduced by employing a CHF process than by supplemental -glucosidase.Offprint requests to: C. M. Hogan     

Examining the social landscapes of alternative reproductive strategies

Social environments are inherently dynamic, often changing depending on the frequency and outcomes of conspecific interactions – they can be simultaneously the targets and agents of selection. Understanding how organisms settle in heterogeneous social environments and the effects this has on reproductive success is vital to our understanding of the selective forces at work in wild populations. From an intensive behavioural and ecological study of territoriality in the polymorphic white‐throated sparrow (Zonotrichia albicollis), we demonstrate that males of the two morphs inhabit social niches that correspond to their respective alternative reproductive strategies. By integrating molecular ecology and Geographic Information Systems, we generated continuous, socio‐spatial models of local conspecific density and cuckoldry risk. Our results suggest that the morphs segregate their territories based on socio‐spatial variables, creating a heterogeneous social landscape that matches each behavioural phenotype with a favourable social environment. Specifically, the monogamous tan males tended to settle in low‐density areas that were also low for cuckoldry risk, while the opposite was true for the promiscuous white males. This pattern of socio‐spatial heterogeneity, combined with the social niche partitioning we observed, might act as a social niche polymorphism, and play an important role in maintenance of the alternative reproductive strategies of the white‐throated sparrow. Socio‐spatial factors, similar to those observed in the white‐throated sparrow, may play important roles in the evolution of mating systems in other species, even those with more continuous or cryptic variation.     

Assessment of ancestry probabilities in the presence of genotyping errors

This paper extends an approach for estimating the ancestry probability, the probability that an inbred line is an ancestor of a given hybrid, to account for genotyping errors. The effect of such errors on ancestry probability estimates is evaluated through simulation. The simulation study shows that if misclassification is ignored, then ancestry probabilities may be slightly overestimated. The sensitivity of ancestry probability calculations to the assumed genotyping error rate is also assessed.     

Differential response to a competitor by Atlantic salmon adopting alternative life-history strategies

The life cycle of the Atlantic salmon is extremely variable. In good growing conditions, juvenile salmon either metamorphose into the migratory phase by their second spring, or delay this for at least another year. The strategy appears to be decided in their first summer. This study compared competitive responses of fish adopting the two strategies. Laboratory experiments showed that the two types of fish had similar foraging efficiencies in isolation. However, although a simulated competitor had little effect on the feeding behaviour of fast-developing fish, it caused an 18-fold increase in the incidence of failed feeding attempts by fish delaying development. The probability of an attack failing was dependent on how close the competitor came.     

An explicit approach to evolutionarily stable dispersal strategies: no cost of dispersal

The evolution of dispersal is examined by looking at evolutionarily stable strategies (ESS) for dispersal parameters in discrete time multisite models without any cost of dispersal. ESS are investigated analytically, based on explicit results on sensitivity analysis of matrix models. The basic model considers an arbitrary number of sites and a single age class. An ESS for dispersal parameters is obtained when the spatial reproductive values, calculated at the density-dependent population equilibrium, are equal across sites. From this basic formulation, one derives equivalently that all local populations should be at equilibrium in the absence of migration, and that dispersal between sites should be balanced, i.e., the numbers of individuals arriving to and leaving a site are equal. These results are then generalized to a model with several age classes. Equal age-specific reproductive values do not however imply balanced dispersal in this case. Our results generalize to any number of sites and age classes those available ?M. Doebeli, Dispersal and dynamics, Theoret. Popul. 47 (1995) 82 for two sites and one age class.