Reduction of Ethanol Yield and Improvement of Glycerol Formation by Adaptive Evolution of the Wine Yeast Saccharomyces cerevisiae under Hyperosmotic Conditions

There is a strong demand from the wine industry for methodologies to reduce the alcohol content of wine without compromising wine''s sensory characteristics. We assessed the potential of adaptive laboratory evolution strategies under hyperosmotic stress for generation of Saccharomyces cerevisiae wine yeast strains with enhanced glycerol and reduced ethanol yields. Experimental evolution on KCl resulted, after 200 generations, in strains that had higher glycerol and lower ethanol production than the ancestral strain. This major metabolic shift was accompanied by reduced fermentative capacities, suggesting a trade-off between high glycerol production and fermentation rate. Several evolved strains retaining good fermentation performance were selected. These strains produced more succinate and 2,3-butanediol than the ancestral strain and did not accumulate undesirable organoleptic compounds, such as acetate, acetaldehyde, or acetoin. They survived better under osmotic stress and glucose starvation conditions than the ancestral strain, suggesting that the forces that drove the redirection of carbon fluxes involved a combination of osmotic and salt stresses and carbon limitation. To further decrease the ethanol yield, a breeding strategy was used, generating intrastrain hybrids that produced more glycerol than the evolved strain. Pilot-scale fermentation on Syrah using evolved and hybrid strains produced wine with 0.6% (vol/vol) and 1.3% (vol/vol) less ethanol, more glycerol and 2,3-butanediol, and less acetate than the ancestral strain. This work demonstrates that the combination of adaptive evolution and breeding is a valuable alternative to rational design for remodeling the yeast metabolic network.     

Trans‐species synthetic gene design allows resistance pyramiding and broad‐spectrum engineering of virus resistance in plants

To infect plants, viruses rely heavily on their host's machinery. Plant genetic resistances based on host factor modifications can be found among existing natural variability and are widely used for some but not all crops. While biotechnology can supply for the lack of natural resistance alleles, new strategies need to be developed to increase resistance spectra and durability without impairing plant development. Here, we assess how the targeted allele modification of the Arabidopsis thaliana translation initiation factor eIF4E1 can lead to broad and efficient resistance to the major group of potyviruses. A synthetic Arabidopsis thaliana eIF4E1 allele was designed by introducing multiple amino acid changes associated with resistance to potyvirus in naturally occurring Pisum sativum alleles. This new allele encodes a functional protein while maintaining plant resistance to a potyvirus isolate that usually hijacks eIF4E1. Due to its biological functionality, this synthetic allele allows, at no developmental cost, the pyramiding of resistances to potyviruses that selectively use the two major translation initiation factors, eIF4E1 or its isoform eIFiso4E. Moreover, this combination extends the resistance spectrum to potyvirus isolates for which no efficient resistance has so far been found, including resistance‐breaking isolates and an unrelated virus belonging to the Luteoviridae family. This study is a proof‐of‐concept for the efficiency of gene engineering combined with knowledge of natural variation to generate trans‐species virus resistance at no developmental cost to the plant. This has implications for breeding of crops with broad‐spectrum and high durability resistance using recent genome editing techniques.     

Rapid development of gene-tagged microsatellite markers from bacterial artificial chromosome clones using anchored TAA repeat primers

We developed a technique to improve the efficiency of producing TAA repeat microsatellite markers linked to interspecific conserved genes. Template DNA was prepared from cultures derived from single bacterial artificial chromosome (BAC) colonies using a simple alkaline lysis miniprep. The presence of conserved genes in each BAC clone was verified by sequencing with gene-specific primers. The BAC templates were directly sequenced using short tandem repeat-anchored primers (STRAPs), consisting of TAA repeats with one or two unique 3' terminal bases. At least one STRAP provided sufficient 3' flanking sequence from each clone for the design of a BAC-specific primer. The BAC-specific primer was used to sequence back through the tandem repeat and obtain 5' flanking sequence, and a second BAC-specific primer was designed for microsatellite genotype analysis. This technique quickly provided microsatellite markers with an average of 15 tandem repeats for the BAC clones tested. The identification of polymorphic microsatellite loci in these clones permits the identification of alleles linked to candidate genes, placement of conserved genes on genetic linkage maps, and integration of linkage and physical maps.     

New preparation techniques for molecular and in‐situ analysis of ancient organic micro‐ and nanostructures

Organic microfossils preserved in three dimensions in transparent mineral matrices such as cherts/quartzites, phosphates, or carbonates are best studied in petrographic thin sections. Moreover, microscale mass spectrometry techniques commonly require flat, polished surfaces to minimize analytical bias. However, contamination by epoxy resin in traditional petrographic sections is problematic for the geochemical study of the kerogen in these microfossils and more generally for the in situ analysis of fossil organic matter. Here, we show that epoxy contamination has a molecular signature that is difficult to distinguish from kerogen with time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). This contamination appears pervasive in organic microstructures embedded in micro‐ to nano‐crystalline carbonate. To solve this problem, a new semi‐thin section preparation protocol without resin medium was developed for micro‐ to nanoscale in situ investigation of insoluble organic matter. We show that these sections are suited for microscopic observation of Proterozoic microfossils in cherts. ToF‐SIMS reveals that these sections are free of pollution after final removal of a <10 nm layer of contamination using low‐dose ion sputtering. ToF‐SIMS maps of fragments from aliphatic and aromatic molecules and organic sulfur are correlated with the spatial distribution of organic microlaminae in a Jurassic stromatolite. Hydrocarbon‐derived ions also appeared correlated with kerogenous microstructures in Archean cherts. These developments in analytical procedures should help future investigations of organic matter and in particular, microfossils, by allowing the spatial correlation of microscopy, spectroscopy, precise isotopic microanalyses, and novel molecular microanalyses such as ToF‐SIMS.     

Potential role of HER2-overexpressing exosomes in countering trastuzumab-based therapy

Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness.     

A Pragmatic Approach to Assess the Exposure of the Honey Bee (Apis mellifera) When Subjected to Pesticide Spray

Plant protection spray treatments may expose non-target organisms to pesticides. In the pesticide registration procedure, the honey bee represents one of the non-target model species for which the risk posed by pesticides must be assessed on the basis of the hazard quotient (HQ). The HQ is defined as the ratio between environmental exposure and toxicity. For the honey bee, the HQ calculation is not consistent because it corresponds to the ratio between the pesticide field rate (in mass of pesticide/ha) and LD₅₀ (in mass of pesticide/bee). Thus, in contrast to all other species, the HQ can only be interpreted empirically because it corresponds to a number of bees/ha. This type of HQ calculation is due to the difficulty in transforming pesticide field rates into doses to which bees are exposed. In this study, we used a pragmatic approach to determine the apparent exposure surface area of honey bees submitted to pesticide treatments by spraying with a Potter-type tower. The doses received by the bees were quantified by very efficient chemical analyses, which enabled us to determine an apparent surface area of 1.05 cm²/bee. The apparent surface area was used to calculate the exposure levels of bees submitted to pesticide sprays and then to revisit the HQ ratios with a calculation mode similar to that used for all other living species. X-tomography was used to assess the physical surface area of a bee, which was 3.27 cm²/bee, and showed that the apparent exposure surface was not overestimated. The control experiments showed that the toxicity induced by doses calculated with the exposure surface area was similar to that induced by treatments according to the European testing procedure. This new approach to measure risk is more accurate and could become a tool to aid the decision-making process in the risk assessment of pesticides.     

Impact of demography on extinction/fixation events

In this article we consider diffusion processes modeling the dynamics of multiple allelic proportions (with fixed and varying population size). We are interested in the way alleles extinctions and fixations occur. We first prove that for the Wright–Fisher diffusion process with selection, alleles get extinct successively (and not simultaneously), until the fixation of one last allele. Then we introduce a very general model with selection, competition and Mendelian reproduction, derived from the rescaling of a discrete individual-based dynamics. This multi-dimensional diffusion process describes the dynamics of the population size as well as the proportion of each type in the population. We prove first that alleles extinctions occur successively and second that depending on population size dynamics near extinction, fixation can occur either before extinction almost surely, or not. The proofs of these different results rely on stochastic time changes, integrability of one-dimensional diffusion processes paths and multi-dimensional Girsanov’s tranform.