Modulating non-native aggregation and electrostatic protein-protein interactions with computationally designed single-point mutations.

Non-native protein aggregation is a ubiquitous challenge in the production, storage and administration of protein-based biotherapeutics. This study focuses on altering electrostatic protein-protein interactions as a strategy to modulate aggregation propensity in terms of temperature-dependent aggregation rates, using single-charge variants of human γ-D crystallin. Molecular models were combined to predict amino acid substitutions that would modulate protein-protein interactions with minimal effects on conformational stability. Experimental protein-protein interactions were quantified by the Kirkwood-Buff integrals (G22) from laser scattering, and G22 showed semi-quantitative agreement with model predictions. Experimental initial-rates for aggregation showed that increased (decreased) repulsive interactions led to significantly increased (decreased) aggregation resistance, even based solely on single-point mutations. However, in the case of a particular amino acid (E17), the aggregation mechanism was altered by substitution with R or K, and this greatly mitigated improvements in aggregation resistance. The results illustrate that predictions based on native protein-protein interactions can provide a useful design target for engineering aggregation resistance; however, this approach needs to be balanced with consideration of how mutations can impact aggregation mechanisms.

Development of at-line assay to monitor charge variants of MAbs during production.

One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro-heterogeneity can result from post-translational modifications such as sialylation, galactosylation, C-terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on-line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired post-translational modifications and PK behavior. However, one major restriction to implementation of quality control strategies is the availability of techniques for obtaining on-line or at-line measurements of these attributes. In this work, we describe the development of an at-line assay to separate MAb charge variants in near real-time, which could ultimately be used to implement on-line quality control strategies for MAb production. The assay consists of a 2D-HPLC method with sequential in-line Protein A and WCX-10 HPLC column steps. To perform the 2D-HPLC assay at-line, the two columns steps were integrated into a single method using a novel system configuration that allowed parallel flow over column 1 or column 2 or sequential flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and delivered to the 2D-HPLC for analysis. With this at-line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions.

Transgenic mouse and cell culture models demonstrate a lack of mechanistic connection between endoplasmic reticulum stress and tau dysfunction.

In vivo aggregation of tau protein is a hallmark of many neurodegenerative disorders, including Alzheimer's disease (AD). Recent evidence has also demonstrated activation of the unfolded protein response (UPR), a cellular response to endoplasmic reticulum (ER) stress, in AD, although the role of the UPR in disease pathogenesis is not known. Here, three model systems were used to determine whether a direct mechanistic link could be demonstrated between tau aggregation and the UPR. The first model system used was SH-SY5Y cells, a neuronal cultured cell line that endogenously expresses tau. In this system, the UPR was activated using chemical stressors, tunicamycin and thapsigargin, but no changes in tau expression levels, solubility, or phosphorylation were observed. In the second model system, wild-type 4R tau and P301L tau, a variant with increased aggregation propensity, were heterologously overexpressed in HEK 293 cells. This overexpression did not activate the UPR. The last model system examined here was the PS19 transgenic mouse model. Although PS19 mice, which express the P301S variant of tau, display severe neurodegeneration and formation of tau aggregates, brain tissue samples did not show any activation of the UPR. Taken together, the results from these three model systems suggest that a direct mechanistic link does not exist between tau aggregation and the UPR.

Enabling technologies to improve area-wide integrated pest management programmes for the control of screwworms.

The economic devastation caused in the past by the New World screwworm fly Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) to the livestock industry in the U.S.A., Mexico and the rest of Central America was staggering. The eradication of this major livestock pest from North and Central America using the sterile insect technique (SIT) as part of an area-wide integrated pest management (AW-IPM) programme was a phenomenal technical and managerial accomplishment with enormous economic implications. The area is maintained screwworm-free by the weekly release of 40 million sterile flies in the Darien Gap in Panama, which prevents migration from screwworm-infested areas in Columbia. However, the species is still a major pest in many areas of the Caribbean and South America and there is considerable interest in extending the eradication programme to these countries. Understanding New World screwworm fly populations in the Caribbean and South America, which represent a continuous threat to the screwworm-free areas of Central America and the U.S.A., is a prerequisite to any future eradication campaigns. The Old World screwworm fly Chrysomya bezziana Villeneuve (Diptera: Calliphoridae) has a very wide distribution ranging from Southern Africa to Papua New Guinea and, although its economic importance is assumed to be less than that of its New World counterpart, it is a serious pest in extensive livestock production and a constant threat to pest-free areas such as Australia. In the 1980s repeated introductions and an expansion of Old World screwworm populations were reported in the Middle East; in the 1990s it invaded Iraq and since late 2007 it has been reported in Yemen, where a severe outbreak of myiasis occurred in 2008. Small-scale field trials have shown the potential of integrating the SIT in the control of this pest and various international organizations are considering using the release of sterile insects as part of an AW-IPM approach on a much wider scale. Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae) is a screwworm of temperate regions, which, although of limited agricultural importance, has invaded several new locations in the past few years. This special issue reports on the results of a 6-year project funded by the Joint Food and Agriculture Organization of the United Nations/International Atomic Energy Agency (FAO/IAEA) Programme of Nuclear Techniques in Food and Agriculture entitled 'Enabling Technologies for the Expansion of the SIT for Old and New World Screwworm'. A major goal of the project was to better understand population genetic variation in screwworms as an aid to the identification of isolated populations. The project also addressed issues related to genetic sexing, cuticular hydrocarbons, population dynamics, genetic transformation and chromosome analysis.

Kinetic folding studies of the P22 tailspike beta-helix domain reveal multiple unfolded states.

The beta-helix is an important protein fold in many pathogens, and is a challenging system for folding pathway prediction because it primarily is stabilized by non-local interactions along the primary sequence. A useful experimental model of this fold is a monomeric truncation of P22 tailspike protein, the beta-helix domain (bhx). This report describes a systematic in vitro study of the chemical denaturation and refolding of bhx. Results from equilibrium chemical denaturation experiments were consistent with a two-state folding mechanism, but showed only partial reversibility. Stopped-flow fluorescence studies showed a single unfolding step, but two refolding steps. The slow refolding step could be partly attributed to proline isomerization, based on an increased rate during refolding in the presence of PPIase and an increased relative amplitude of this step with increasing delay time in double-jump refolding experiments observed over delays of 5-100 s. However, double-jump refolding experiments with delay times longer than 100 s along with size exclusion chromatography and dynamic light scattering of refolding samples showed that the overall refolding yield decreased as bhx was unfolded for longer periods of time. Furthermore, the losses resulted from aggregate formation during refolding. This suggests that a change occurs over time in the unfolded or denatured state ensemble that increases the propensity for aggregation upon the shift to more native-favoring conditions. Alternatively aggregate nuclei may be able to form even under high denaturant conditions, and these subsequently grow and consume monomer when placed under native-favoring conditions.

Mitotic and polytene chromosome analysis in the Mexican fruit fly, Anastrepha ludens (Loew) (Diptera: Tephritidae).

The present study constitutes the first attempt to construct a polytene chromosome map of an Anastrepha species, Anastrepha ludens (Loew), a major agricultural pest. The mitotic karyotype has a diploid complement of 12 acrocentric chromosomes, including five pairs of autosomes and an XX/XY sex chromosome pair. The analysis of salivary gland polytene chromosomes has shown a total number of five polytene elements that correspond to the five autosomes. The characteristic features and the most prominent landmarks of each chromosome are described. By comparing chromosome banding patterns, the possible chromosomal homology between A. ludens and Ceratitis capitata (Wiedemann) is presented. This work shows that polytene maps of A. ludens are suitable for cytogenetic studies in this species and may be used as reference for other Anastrepha species, most of which are also serious agricultural pests.

Molecular phylogenetics of tsetse flies (Diptera: Glossinidae) based on mitochondrial (COI, 16S, ND2) and nuclear ribosomal DNA sequences, with an emphasis on the palpalis group.

Relationships of 13 species of the genus Glossina (tsetse flies) were inferred from mitochondrial (cytochrome oxidase 1, NADH dehydrogenase 2 and 16S) and nuclear (internal transcribed spacer 1 of rDNA) sequences. The resulting phylogeny confirms the monophyly of the morphologically defined fusca, morsitans and palpalis subgenera. Genetic distances between palpalis and morsitans subspecies suggest that their status needs revision. In particular, cytochrome oxidase 1 sequences showed large geographical differences within G. palpalis palpalis, suggesting the existence of cryptic species within this subspecies. The morphology of palpalis group female genital plates was examined, and individuals were found varying outside the ranges specified by the standard identification keys, making definitive morphological classification impossible. A diagnostic PCR to distinguish G. palpalis palpalis, G. tachinoides and G. palpalis gambiensis based on length differences of internal transcribed spacer 1 sequences is presented.

Polymorphic microsatellite markers for the tsetse fly Glossina fuscipes fuscipes (Diptera: Glossinidae), a vector of human African trypanosomiasis.

Our understanding of Glossina fuscipes fuscipes, a major vector of sleeping sickness, has been severely constrained by a lack of genetic markers for mapping and population genetic studies. Here we present 10 newly developed microsatellite loci for this tsetse species. Heterozygosity levels in Moyo, an Ugandan population, averaged 0.57, with only two loci showing very low heterozygosity. Five loci carried more than six alleles. Together with five recently published microsatellite loci, this brings the number of available microsatellite loci for this species to 15. Their availability will greatly facilitate future studies on the genetics of this important human disease vector.

Copulation behaviour of Glossina pallidipes (Diptera: Muscidae) outside and inside the female, with a discussion of genitalic evolution.

If species-specific male genitalia are courtship devices under sexual selection by cryptic female choice, then species-specific aspects of the morphology and behaviour of male genitalia should often function to stimulate the female during copulation. The morphology and behaviour of the complex, species-specific male genitalia of the tsetse fly, Glossina pallidipes Austen, were determined from both direct observations and dissections of flash-frozen copulating pairs; we found that some male genitalic traits probably function to stimulate the female, while others function to restrain her. The male clamps the ventral surface of the female's abdomen tightly with his powerful cerci. Clamping does not always result in intromission. Clamping bends the female's body wall and her internal reproductive tract sharply, posteriorly and dorsally, and pinches them tightly. The male performed sustained, complex, stereotyped, rhythmic squeezing movements with his cerci that were not necessary to mechanically restrain the female and appeared instead to have a stimulatory function. Six different groups of modified setae on and near the male's genitalia rub directly against particular sites on the female during squeezing. The designs of these setae correlate with the force with which they press on the female and the probable sensitivity of the female surfaces that they contact. As expected under the hypothesis that these structures are under sexual selection by female choice, several traits suspected to have stimulatory functions have diverged in G. pallidipes and its close relative, G. longipalpis. Additional male non-genitalic behaviour during copulation, redescribed more precisely than in previous publications, is also likely to have a courtship function. The elaborate copulatory courtship behaviour and male genitalia may provide the stimuli that previous studies showed to induce female ovulation and resistance to remating.

Polytene chromosome maps in four species of tsetse flies Glossina austeni, G. pallidipes, G. morsitans morsitans and G. m. submorsitans (Diptera: Glossinidae): a comparative analysis.

Photographic polytene chromosome maps from pupal trichogen cells of four tsetse species, Glossina austeni, G. pallidipes, G. morsitans morsitans and G. m. submorsitans were constructed and compared. The homology of chromosomal elements between the species was achieved by comparing banding patterns. The telomeric and subtelomeric chromosome regions were found to be identical in all species. The pericentromeric regions were found to be similar in the X chromosome and the left arm of L1 chromosome (L1L) but different in L2 chromosome and the right arm of L1 chromosome (L1R). The L2 chromosome differs by a pericentric inversion that is fixed in the three species, G. pallidipes, G. morsitans morsitans and G. m. submorsitans. Moreover, the two morsitans subspecies appeared to be homosequential and differ only by two paracentric inversions on XL and L2L arm. Although a degree of similarity was observed across the homologous chromosomes in the four species, the relative position of specific chromosome regions was different due to chromosome inversions established during their phylogeny. However, there are regions that show no apparent homology between the species, an observation that may be attributed to the considerable intra--chromosomal rearrangements that have occurred following the species divergence. The results of this comparative analysis support the current phylogenetic relationships of the genus Glossina.

Sex separation of tsetse fly pupae using near-infrared spectroscopy.

Implementation of the sterile insect technique for tsetse (Glossina spp.) requires that only sterile male insects be released; thus, at some stage of the fly production process the females have to be removed. A further constraint in the use of the sterile insect technique for tsetse is that the females are needed for colony production and hence, a non-destructive method of sex separation is required. In most tsetse sterile insect technique programmes thus far, females have been eliminated from the released material by hand-separation of chilled adults. Using near-infrared (NIR) spectroscopy, significant differences have been found between the spectra for the pupae of male and female G. pallidipes Austen. Significantly, the differences appear to be maximized 4-5 days before emergence of the adults. Tsetse fly pupae up to five days before emergence can be sexed with accuracies that generally range from 80 to 100%. This system, when refined, will enable effective separation of male and female pupae to be carried out, with emerged females being returned to the colony and males being irradiated and released. If separation can be achieved five days before emergence, this will also enable irradiated male pupae to be shipped to other destinations as required. Other Diptera were evaluated using this system but had lower classification accuracies of 50-74%. This may be due to the difference in reproductive physiology between these different fly groups.

Analysis of cellular response to protein overexpression.

The overexpression of secreted proteins is of critical importance to the biotechnology and biomedical fields. A common roadblock to high yields of proteins is in the endoplasmic reticulum (ER) where proofreading for properly folded proteins is often rate limiting. Heterologous expression of secreted proteins can saturate the cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. An obvious method for overcoming this block would be to increase the capacity of the folding process (overexpressing chaperones) or decreasing the proofreading process (blocking the down-regulation by the UPR). Unfortunately, these processes are tightly interlinked, whereby modification of one mechanism has unknown effects on the other. Although some success has been achieved in improving expression via co-overexpressing ER chaperones, the results have not lead to a global method for increasing all heterologously overexpressed proteins. Further, many diseases have been linked to extended periods of stress and are not treatable by these approaches. This work utilises both experimental analysis of the interactions within the ER and modelling in order to understand how these interactions affect early secretory pathway dynamics. This study shows that overexpression of the ER chaperone binding protein does not regulate Ire1p and the UPR as predicted by a model based on the published understanding of the molecular mechanism. A new model is proposed for Ire1p regulation and the UPR that better fits the experimental data and recent studies on Ire1p.

Mitochondrial diversity analysis of Glossina palpalis gambiensis from Mali and Senegal.

West African riverine tsetse populations of Glossina palpalis gambiensis Vanderplank (Diptera: Glossinidae) were investigated for gene flow, inferred from mitochondrial diversity in samples of 69 flies from Senegal and 303 flies from three river drainages in Mali. Four polymorphic mitochondrial loci were scored. Mean haplotype diversities were 0.30 in Mali, 0 in Senegal and 0.18 over both Mali and Senegal. These diversities estimate the probabilities that two randomly chosen tsetse have different haplotypes. Substantial rates of gene flow were detected among flies sampled along tributaries belonging to the river basins of the Senegal, Niger, and Bani in Mali. There was virtually no gene flow between tsetse in Senegal and Mali. No seasonal effects on gene flow were detected. The implications of these preliminary findings for the implementation of area-wide integrated pest management (AW-IPM) programmes against riverine tsetse in West Africa are discussed.

P22 tailspike trimer assembly is governed by interchain redox associations.

Though disulfide bonds are absent from P22 tailspike protein in its native state, a disulfide-bonded trimeric intermediate has been identified in the tailspike folding and assembly pathway in vitro. The formation of disulfide bonds is critical to efficient assembly of native trimers as mutations at C-terminal cysteines reduce or inhibit trimer formation. We investigated the effect of different redox folding environments on tailspike formation to discover if simple changes in reducing potential would facilitate trimer formation. Expression of tailspike in trxB cell lines with more oxidizing cytoplasms led to lower trimer yields; however, observed assembly rates were unchanged. In vitro, the presence of any redox buffer decreased the overall yield compared to non-redox buffered controls; however, the greatest yields of the native trimer were obtained in reducing rather than oxidizing environments at pH 7. Slightly faster trimer formation rates were observed in the redox samples at pH 7, perhaps by accelerating the reduction of the disulfide-bonded protrimer to the native trimer. These rates and the effects of the redox system were found to depend greatly on the pH of the refolding reaction. Oxidized glutathione (GSSG) trapped a tailspike intermediate, likely as a mixed disulfide. This trapped intermediate was able to form native trimer upon addition of dithiothreitol (DTT), indicating that the trapped intermediate is on the assembly pathway, rather than the aggregation pathway. Thus, the presence of redox agents interfered with the ability of the tailspike monomers to associate, demonstrating that disulfide associations play an important role during the assembly of this cytoplasmic protein.

The effect of age on the mating competitiveness of male Glossina fuscipes fuscipes and G. palpalis palpalis.

The effect of age on male Glossina fuscipes fuscipes, Newstead, and Glossina palpalis palpalis, Austin (Diptera: Glossinidae) competiveness were investigated with a view to estimate optimal age for sterile male release. Sterile insect technique involves the mass production, sterilization and sequential release of males of the target species to out compete the wild male population. Mating between released sterile males and wild females produce inviable progeny and the population is reduced over several generations to unsustainable levels. It is vital that the released male are of high quality and are sexually competitive. Age is one parameter affecting the sexual competiveness of the male tsetse fly. The optimal release age was estimated by assessing sexual competitiveness of flies of different age categories, 1, 5, 8 and 13-days after adult eclosion. A walk-in field-cage was used in order to approximate as closely as possible the actual field scenario during sterile insect release programes. It was shown that 8 and 13-day old males mated significantly more frequently, i.e. were more competitive, in the presence of equal numbers of 1 and 5-day old males. The age of male tsetse flies significantly affected competitiveness in both species studied. The ability of G. f. fuscipes to inseminate was not age dependent, and insemination occurred in all females that mated regardless of male age. In G. p. palpalis, however, 1-day old males were least able to inseminate. Mating duration was not significantly affected by age in both species. Eight to thirteen day old males of the test species are here recommended as the optimal sterile male release age.

Genetic sexing strains in medfly, Ceratitis capitata, sterile insect technique programmes.

The introduction of genetic sexing strains (GSS) into medfly, Ceratitis capitata (Wiedemann), sterile insect technique (SIT) programmes started in 1994 and it was accompanied by extensive evaluation of the strains both in field cages and in open field situations. Two male-linked translocation systems, one based on pupal colour, wp, and the other based on temperature sensitivity, tsl, have been used in medfly SIT programmes and they have quite different impacts on mass rearing strategy. In strains based on tsl, female zygotes are killed using high temperature and for wp strains, female and male pupae are separated based on their colour. In all these systems the colony females are homozygous for the mutation requiring that the mutation is not too deleterious and the males are also semi-sterile due to the presence of a male-linked translocation. Managing strain stability during large-scale mass rearing has presented some problems that have been essentially solved by selecting particular translocations for GSS and by the introduction of a filter rearing system (FRS). The FRS operates by removing from the colony any recombinant individuals that threaten the integrity of the strain. The use of GSS opens up the possibility of using the SIT for suppression as opposed to eradication and different radiation strategies can be considered. Some of the many field trials of the strains that were carried out before the strains were introduced into operational programmes are reviewed and an overview is given of their current use.

Assessing mating performance of male Glossina pallidipes (Diptera: Glossinidae) using a walk-in field cage.

To monitor the quality of male tsetse for use in the sterile insect technique (SIT), a field cage test was developed and evaluated. Mating competitiveness was tested with male Glossina pallidipes Austen that emerged from pupae stored for different periods at 15 degrees C. Control males emerged from pupae stored at 23-24 degrees C and emerged at 26.5 degrees C. Each sample of test males was divided into two groups with one group being irradiated at 120 Gy; the other group was not irradiated. More than 70% of the maximum possible number of mating pairs occurred in all tests. Males emerged from pupae kept at low temperature and then irradiated formed a greater proportion of mating pairs than the controls. Males emerged from pupae kept at 15 degrees C generally started mating more quickly than the standard colony males although there was no significant difference. Insemination rates were above 99%. Pooled data indicated that mean spermathecal values for females mated with irradiated males were significantly lower than for control males. The duration of copulation varied significantly between treatment groups and was significantly longer for irradiated male flies; there was no correlation between duration of copulation and mean spermathecal value.

Hermes-mediated germ-line transformation of the Mediterranean fruit fly Ceratitis capitata.

We report the use of the Hermes transposable element for germ-line transformation of the Mediterranean fruit fly, Ceratitis capitata. Hermes was able to genetically transform this insect at an estimated frequency between 0.6 and 1.1%, which is comparable to the transformation frequencies obtained for this species when using other transposable elements. Hermes integrates into the medfly genome by a cut-and-paste mechanism and the sequences integrated into the genome are delimited by the terminal nucleotides of the Hermes inverted terminal repeats. Integration resulted in the generation of 8 bp target site duplications, the sequences of which conformed to the target site duplications generated by hAT element transposition in insects. The Hermes element is one additional genetic tool that can be deployed in manipulating and characterizing the medfly genome.

Microsatellite polymorphism in tsetse flies (Diptera: Glossinidae).

In sub-Saharan Africa, tsetse flies are the vectors of trypanosomes, the causative agents of sleeping sickness in humans and nagana in animals. Certain wild populations of the palpalis group exhibit intraspecific variation and are suspect of manifest differences in vectorial capacity. The current study reports the identification of 13 polymorphic microsatellite loci from Glossina palpalis palpalis Robinean-Desvoidy. The majority of these markers amplify corresponding loci from the related species C. p. gambiensis Vanderplank, G. f. fuscipes Newstead, and G. tachinoides Westwood. Only seven of 13 loci were amplified from G. austeni Newstead. Genetic variability was estimated in one field population of G. p. gambiensis. These results confirmed that microsatellite markers may be used to examine the subpopulation structure of tsetse flies.

Prospects for control of African trypanosomiasis by tsetse vector manipulation.

The extensive antigenic variation phenomena African trypanosomes display in their mammalian host have hampered efforts to develop effective vaccines against trypanosomiasis. Human disease management aims largely to treat infected hosts by chemotherapy, whereas control of animal diseases relies on reducing tsetse populations as well as on drug therapy. The control strategies for animal diseases are carried out and financed by livestock owners, who have an obvious economic incentive. Sustaining largely insecticide-based control at a local level and relying on drugs for treatment of infected hosts for a disease for which there is no evidence of acquired immunity could prove extremely costly in the long run. It is more likely that a combination of several methods in an integrated, phased and area-wide approach would be more effective in controlling these diseases and subsequently improving agricultural output. New approaches that are environmentally acceptable, efficacious and affordable are clearly desirable for control of various medically and agriculturally important insects including tsetse. Here, Serap Aksoy and colleagues discuss molecular genetic approaches to modulate tsetse vector competence.