Experimental evolution of a pheromone signal

Sexual signals are important in speciation, but understanding their evolution is complex as these signals are often composed of multiple, genetically interdependent components. To understand how signals evolve, we thus need to consider selection responses in multiple components and account for the genetic correlations among components. One intriguing possibility is that selection changes the genetic covariance structure of a multicomponent signal in a way that facilitates a response to selection. However, this hypothesis remains largely untested empirically. In this study, we investigate the evolutionary response of the multicomponent female sex pheromone blend of the moth Heliothis subflexa to 10 generations of artificial selection. We observed a selection response of about three‐quarters of a phenotypic standard deviation in the components under selection. Interestingly, other pheromone components that are biochemically and genetically linked to the components under selection did not change. We also found that after the onset of selection, the genetic covariance structure diverged, resulting in the disassociation of components under selection and components not under selection across the first two genetic principle components. Our findings provide rare empirical support for an intriguing mechanism by which a sexual signal can respond to selection without possible constraints from indirect selection responses.     

Multilocus definition of MHC haplotypes in pedigreed cynomolgus macaques (Macaca fascicularis)

Cynomolgus macaques (Macaca fascicularis) are used widely in biomedical research, and the genetics of their MHC (Mhc-Mafa) has become the focus of considerable attention in recent years. The cohort of Indonesian pedigreed macaques that we present here was typed for Mafa-A, -B, and -DR, by sequencing, as described in earlier studies. Additionally, the DRB region of these animals was characterised by microsatellite analyses. In this study, full-length sequencing of Mafa-DPA/B and -DQA/B in these animals was performed. A total of 75 different alleles were observed; 22 of which have not previously been reported, plus 18 extended exon 2 alleles that were already known. Furthermore, two microsatellites, D6S2854 and D6S2859, were used to characterise the complex Mafa-A region. Sequencing and segregation analyses revealed that the length patterns of these microsatellites are unique for each Mafa-A haplotype. In this work, we present a pedigreed colony of approximately 120 cynomolgus macaques; all of which are typed for the most significant polymorphic MHC class I and class II markers. Offspring of these pedigreed animals are easily characterised for their MHC by microsatellite analyses on the Mafa-A and -DRB regions, which makes the cumbersome sequencing analyses redundant.     

Reduction of chronic graft injury with a new HTK-based preservation solution in a murine heart transplantation model

Objective

Aim of this study was to evaluate a new histidine-tryptophan-ketoglutarate (HTK)-based preservation solution on chronic isograft injury in comparison to traditional HTK solution.

Methods

Hearts of C57BL/6J (H-2b) mice were stored for 15 h in 0–4 °C cold preservation solution and then transplanted heterotopically into C57BL/6J (H-2b) mice. Three groups were evaluated: HTK, the base solution of a new preservation solution and hearts without cold ischemia (control). Time to restoration of heartbeat was measured (re-beating time). Strength of the heartbeat was palpated daily and scored on a 4-level scale (palpation score). Animals were sacrificed after 60 days of observation (24 h for TGF-β expression). The transplanted hearts were evaluated histologically for myocardial damage, vasculopathy and interstitial fibrosis. TGF-β expression was assessed immunohistologically. All investigators were blinded to the groups. ANOVA and LSD post hoc test were used for statistical analysis.

Results

The re-beating time was significantly shorter in hearts stored in the new solution (10.3 ± 2.6 min vs. HTK 14.2 ± 4.1 min; p < 0.05). The palpation score was significantly higher in hearts stored in the new solution (2.3 ± 0.4 vs. HTK 1.6 ± 0.5; p < 0.01). Hearts stored in the new solution showed a lower myocardial injury score (1.8 ± 0.2 vs. HTK 2.2 ± 0.7), less interstitial fibrosis (4.8 ± 1.9% vs. HTK 8.5 ± 3.8%, p < 0.05), less vasculopathy (14.7 ± 6.9% vs. 22.0 ± 23.2%; p = 0.06) and lower TGF-β1-expression (6.6 ± 1.4% vs. HTK 12.0 ± 4.6%).

Conclusion

The new HTK-based solution reduces the chronic isograft injury. This protective effect is likely achieved through several modifications and supplements into the new solution like N-acetyl-l-histidine, glycine, alanine, arginine and sucrose.     

The influence of aerated hydration seed treatment on seed longevity as assessed by the viability equations

Aerated hydration (AH) treatments of cauliflower seeds for 12 h (12AH) and 28 h (28AH) at 20 degrees C resulted in improved or reduced storage potential of low or high vigour seeds, respectively. Seeds were stored at their initial seed moisture content (mean 5.5% mc) or at 12% mc at 10 degrees C for 12 months and at 20 degrees C for 4 months. The improved longevity of low vigour seeds was associated with increased K(i) (initial seed viability) and a reduced rate of deterioration (1/sigma) whereas the K(i) of high vigour seeds fell after 28AH and the rate of deterioration increased such that the time to lose one probit of viability decreased from 28.7 to 5.3 months at 10 degrees C and from 10.4 to 1.2 months at 20 degrees C. The improved K(i) of low vigour seeds could be explained by the reduction in the extent of deterioration after AH, as indicated by the increase in germination after cotrolled deterioration (CD), and the possible activation of metabolic repair during treatment. In contrast the reduced germination after CD of AH-treated high vigour seeds was indicative of deterioration as a result of treatment. Both high and low vigour seeds contained constitutive levels of ss-tubulin which increased during AH treatment, the increase being greater in high vigour seeds. High vigour seeds also showed an increase in the proportion of nuclear DNA present as 4C DNA, from 3% (untreated seeds) to 26% (28AH), indicative of germination advancement from the G(1) to G(2) phase of the cell cycle during treatment. This higher proportion of 4C DNA is correlated with the increased sensitivity of seeds to drying and/or storage after AH, leading to their reduced K(i) and storage potential. In contrast, there was little change in %4C in low vigour seeds. Priming in polyethylene glycol (PEG, -1.0 MPa) for 5 d or 13 d also improved the longevity of low vigour seeds stored at their initial and 12% mc at 10 degrees C for 8 months, as reflected in their laboratory and CD germination. In this case, however, the improved longevity of the low vigour seeds following 13 d priming was associated with an increase in 4C DNA from 4% (dry control) to 56% after treatment. The germination of both untreated and primed high vigour seeds remained high throughout the storage period. Increases in the rate of germination (decreased mean germination time) observed after all AH and PEG treatments were not consistently associated with an increase in the proportion of nuclei containing 4C DNA.     

Seed storage at elevated partial pressure of oxygen, a fast method for analysing seed ageing under dry conditions

Background and Aims

Despite differences in physiology between dry and relative moist seeds, seed ageing tests most often use a temperature and seed moisture level that are higher than during dry storage used in commercial practice and gene banks. This study aimed to test whether seed ageing under dry conditions can be accelerated by storing under high-pressure oxygen.

Methods

Dry barley (Hordeum vulgare), cabbage (Brassica oleracea), lettuce (Lactuca sativa) and soybean (Glycine max) seeds were stored between 2 and 7 weeks in steel tanks under 18 MPa partial pressure of oxygen. Storage under high-pressure nitrogen gas or under ambient air pressure served as controls. The method was compared with storage at 45 °C after equilibration at 85 % relative humidity and long-term storage at the laboratory bench. Germination behaviour, seedling morphology and tocopherol levels were assessed.

Key Results

The ageing of the dry seeds was indeed accelerated by storing under high-pressure oxygen. The morphological ageing symptoms of the stored seeds resembled those observed after ageing under long-term dry storage conditions. Barley appeared more tolerant of this storage treatment compared with lettuce and soybean. Less-mature harvested cabbage seeds were more sensitive, as was the case for primed compared with non-primed lettuce seeds. Under high-pressure oxygen storage the tocopherol levels of dry seeds decreased, in a linear way with the decline in seed germination, but remained unchanged in seeds deteriorated during storage at 45 °C after equilibration at 85 % RH.

Conclusions

Seed storage under high-pressure oxygen offers a novel and relatively fast method to study the physiology and biochemistry of seed ageing at different seed moisture levels and temperatures, including those that are representative of the dry storage conditions as used in gene banks and commercial practice.     

Metabolic control analysis of Aspergillus niger L-arabinose catabolism

A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography, and their kinetic properties were characterized. For the other enzymes of the pathway the kinetic data were available from the literature. The metabolic model was used to analyze flux and metabolite concentration control of the L-arabinose catabolic pathway. The model demonstrated that flux control does not reside at the enzyme following the intermediate with the highest concentration, L-arabitol, but is distributed over the first three steps in the pathway, preceding and following L-arabitol. Flux control appeared to be strongly dependent on the intracellular L-arabinose concentration. At 5 mM intracellular L-arabinose, a level that resulted in realistic intermediate concentrations in the model, flux control coefficients for L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase were 0.68, 0.17 and 0.14, respectively. The analysis can be used as a guide to identify targets for metabolic engineering aiming at either flux or metabolite level optimization of the L-arabinose catabolic pathway of A. niger. Faster L-arabinose utilization may enhance utilization of readily available organic waste containing hemicelluloses to be converted into industrially interesting metabolites or valuable enzymes or proteins.     

Effect of apolipoproteins E and C-III on the interaction of chylomicrons with parenchymal and non-parenchymal cells from rat liver.

[3H]Triacylglycerol-labelled chylomicrons were isolated from intestinal lymph, obtained from rats made hypolipidaemic by treatment with pharmacological amounts of 17 alpha-ethynyloestradiol. Oestrogen treatment results in a large reduction in the content of apolipoproteins (apo) E and C of lymph chylomicrons. Upon incubation in vitro with freshly isolated parenchymal and non-parenchymal cells the apo E-, apo C-poor chylomicrons became readily cell-associated. With increasing chylomicron concentrations this cell-association was saturable and half-maximal cell-association was achieved at about 0.55 mg of triacylglycerol/ml. The cell-association was time- and temperature-dependent. A more than 90% inhibition of the cell-association of the [3H]triacylglycerol moiety was observed with both parenchymal and non-parenchymal cells when pure apo C-III (12.6 micrograms/mg of triacylglycerol) was incorporated into the chylomicrons. These data indicate that apo E-, apo C-poor chylomicrons are bound to both parenchymal and non-parenchymal liver cells at a high-affinity site of limited capacity and that binding to this site is strongly inhibited by apo C-III. With apo C-III-enriched chylomicrons simultaneous determination of the cell-association of the 125I-apo C-III and the [3H]triacylglycerol moiety indicated that more 125I-apo C-III becomes associated to the cells than expected on the basis of [3H]triacylglycerol radioactivity measurements. It is suggested that upon cell-association of apo C-III its binding to the chylomicron particles is lost. Consequently the occupation of the cellular recognition site by apo C-III prevents further chylomicron binding and thus leads to a decrease of the cell-association level of the [3H]triacylglycerol moiety. Apo E enrichment of the chylomicrons led to an increased cell-association rate with parenchymal cells and to a marked increase of the cell-association level with non-parenchymal cells. The cell-association of the apo E radioactivity followed closely the [3H]triacylglycerol radioactivity, indicating that the particle-apo E complex is bound as a unity. The apo E effects were opposed by apo C-III. With apo E-, apo C-III-enriched chylomicrons more 125I-apo E became associated with the cells than could be expected on the basis of the [3H]triacylglycerol measurements. It is concluded that apo C-III can weaken the interaction of apo E with the chylomicrons leading to the cell-association of free apo E. It appears that subtle changes in the apo E and/or apo C-III content of chylomicrons can influence the interaction with both parenchymal and non-parenchymal liver cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic mutation analysis at early stages of cell line development using next generation sequencing

A central goal for most biopharmaceutical companies is to reduce the development timeline to reach clinical proof of concept. This objective requires the development of tools that ensure the quality of biotherapeutic material destined for the clinic. Recent advances in high throughput protein analytics provide confidence in our ability to assess productivity and product quality attributes at early stages of cell line development. However, one quality attribute has, until recently, been absent from the standard battery of analytical tests facilitating informed choices early in cell line selection: genetic sequence confirmation. Techniques historically used for mutation analysis, such as detailed mass spectrometry, have limitations on the sample number and turnaround times making it less attractive at early stages. Thus, we explored the utility of Next‐Generation Sequencing (NGS) as a solution to address these limitations. Amplicon sequencing is one such NGS technique that is robust, rapid, sensitive, and amenable to multiplexing, all of which are essential attributes for our purposes. Here we report a NGS method based upon amplicon sequencing that has been successfully incorporated into our cell line development workflow alongside other high‐throughput protein analytical assays. The NGS method has demonstrated its value by identifying at least one Chinese hamster ovary (CHO) clone expressing a variant form of the biotherapeutic in each of the four clinical programs in which it has been utilized. We believe this sequence confirmation method is essential to safely accelerating the time to clinical proof of concept of biotherapeutics, and guard against delays related to sequence mutations. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:813–817, 2016     

CRISPR-Cas9 mediated gene deletions in lager yeast <Emphasis Type="Italic">Saccharomyces pastorianus</Emphasis>

Background

The ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been domesticated for centuries and is used for the industrial fermentation of lager beer. For yet uncharacterised reasons, this hybrid yeast is far more resilient to genetic alteration than its ancestor S. cerevisiae.

Results

This study reports a new CRISPR-Cas9 method for accurate gene deletion in S. pastorianus. This method combined the Streptococcus pyogenes cas9 gene expressed from either a chromosomal locus or from a mobile genetic element in combination with a plasmid-borne gRNA expression cassette. While the well-established gRNA expression system using the RNA polymerase III dependent SNR52 promoter failed, expression of a gRNA flanked with Hammerhead and Hepatitis Delta Virus ribozymes using the RNA polymerase II dependent TDH3 promoter successfully led to accurate deletion of all four alleles of the SeILV6 gene in strain CBS1483. Furthermore the expression of two ribozyme-flanked gRNAs separated by a 10-bp linker in a polycistronic array successfully led to the simultaneous deletion of SeATF1 and SeATF2, genes located on two separate chromosomes. The expression of this array resulted in the precise deletion of all five and four alleles mediated by homologous recombination in the strains CBS1483 and Weihenstephan 34/70 respectively, demonstrating the multiplexing abilities of this gRNA expression design.

Conclusions

These results firmly established that CRISPR-Cas9 significantly facilitates and accelerates genome editing in S. pastorianus.