Parallel laboratory evolution and rational debugging reveal genomic plasticity to S. cerevisiae synthetic chromosome XIV defects

Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic Saccharomyces cerevisiae genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of S. cerevisiae chromosome XIV as part of the Synthetic Yeast Genome project. In parallel to the use of rational engineering approaches to restore wild-type fitness, we used adaptive laboratory evolution to generate a general growth-defect-suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the synthetic chromosome recombination and modification by loxPsym-mediated evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications. </p

The Role of Direct Presentation by Donor Dendritic Cells in Rejection of Minor Histocompatibility Antigen-Mismatched Skin and Hematopoietic Cell Grafts

Background. The success of transplantation is hampered by rejection of the graft by alloreactive T cells. Donor dendritic cells (DC) have been shown to be required for direct priming of immune responses to antigens from major histocompatibility complex-mismatched grafts. However, for immune responses to major histocompatibility complex-matched, minor histocompatibility (H) antigen mismatched grafts, the magnitude of the T-cell response to directly presented antigens is reduced, and the indirect pathway is more important. Therefore, we aimed to investigate the requirement for donor DC to directly present antigen from minor H antigen mismatched skin and hematopoietic grafts.Methods. Langerhans cell-or conventional (c) DC-depleted skin or hematopoietic cells from male DC-specific diphtheria toxin receptor mice were grafted onto, or injected into, syngeneic female recipients, and survival of the male tissue was compared with nondepleted tissue. Activation of the alloreactive immune response was tracked by the expansion of T cells specific for male HY-derived epitopes.Results. Our data demonstrate that depletion of donor Langerhans cell, dermal cDC, or both from skin grafts prolongs their survival but does not prevent rejection. Extended survival correlates with delayed expansion of HY peptide-specific CD8(+) T cells. In addition, depletion of donor cDC delays rejection of male hematopoietic cells.Conclusions. Our results demonstrate for the first time that direct presentation of minor H antigens by donor DC is required for efficient rejection of skin and hematopoietic grafts by CD8(+) T cells. But, in the absence of donor DC, indirect presentation of minor antigens is sufficient to mediate the response

The Multiplanetary Future of Plant Synthetic Biology

The interest in human space journeys to distant planets and moons has been re-ignited in recent times and there are ongoing plans for sending the first manned missions to Mars in the near future. In addition to generating oxygen, fixing carbon, and recycling waste and water, plants could play a critical role in producing food and biomass feedstock for the microbial manufacture of materials, chemicals, and medicines in long-term interplanetary outposts. However, because life on Earth evolved under the conditions of the terrestrial biosphere, plants will not perform optimally in different planetary habitats. The construction or transportation of plant growth facilities and the availability of resources, such as sunlight and liquid water, may also be limiting factors, and would thus impose additional challenges to efficient farming in an extraterrestrial destination. Using the framework of the forthcoming human missions to Mars, here we discuss a series of bioengineering endeavors that will enable us to take full advantage of plants in the context of a Martian greenhouse. We also propose a roadmap for research on adapting life to Mars and outline our opinion that synthetic biology efforts towards this goal will contribute to solving some of the main agricultural and industrial challenges here on Earth

Conventional dendritic cells are required for the activation of helper-dependent CD8 T cell responses to a model antigen after cutaneous vaccination with lentiviral vectors

Cutaneous vaccination with lentiviral vectors generates systemic CD8 T cell responses that have the potential to eradicate tumors for cancer immunotherapy. However, although s.c. immunization with <1 million lentiviral particles clearly primes cytotoxic T cells, vaccination with much higher doses has routinely been used to define the mechanisms of T cell activation by lentiviral vectors. In particular, experiments to test presentation of lentiviral Ags by dendritic cells (DC) require injection of high viral titers, which may result in aberrant transduction of different DC populations. We exploited inducible murine models of DC depletion to investigate which DC prime the lentiviral response after s.c. immunization with low doses of lentiviral particles. In this article, we demonstrate that conventional DC are required to present Ag to CD8 T cells in draining lymph nodes. Langerhans cells are not required to activate the effector response, and neither Langerhans cells nor plasmacytoid DC are sufficient to prime Ag-specific T cells. Immunization drives the generation of endogenous long-lived memory T cells that can be reactivated to kill Ag-specific targets in the absence of inflammatory challenge. Furthermore, lentiviral vaccination activates expansion of endogenous CD4 Th cells, which are required for the generation of effector CD8 T cells that produce IFN-γ and kill Ag-specific targets. Collectively, we demonstrate that after cutaneous immunization with lentiviral particles, CD4-licensed lymph node conventional DC present Ag to CD8 T cells, resulting in the generation of protective endogenous antitumor immunity that may be effective for cancer immunotherapy.8 page(s

Yeast's balancing act between ethanol and glycerol production in low-alcohol wines

Alcohol is fundamental to the character of wine, yet too much can put a wine off-balance. A wine is regarded to be well balanced if its alcoholic strength, acidity, sweetness, fruitiness and tannin structure complement each other so that no single component dominates on the palate. Balancing a wine's positive fruit flavours with the optimal absolute and relative concentration of alcohol can be surprisingly difficult. Over the past three decades, consumers have increasingly demanded wine with richer and riper fruit flavour profiles. In response, grape and wine producers have extended harvest times to increase grape maturity and enhance the degree of fruit flavours and colour intensity. However, a higher degree of grape maturity results in increased grape sugar concentration, which in turn results in wines with elevated alcohol concentration. On average, the alcohol strength of red wines from many warm wine-producing regions globally rose by about 2% (v/v) during this period. Notwithstanding that many of these 'full-bodied, fruit-forward' wines are well balanced and sought after, there is also a significant consumer market segment that seeks lighter styles with less ethanol-derived 'hotness' on the palate. Consumer-focussed wine producers are developing and implementing several strategies in the vineyard and winery to reduce the alcohol concentration in wines produced from well-ripened grapes. In this context, Saccharomyces cerevisiae wine yeasts have proven to be a pivotal strategy to reduce ethanol formation during the fermentation of grape musts with high sugar content (> 240 g l⁻¹). One of the approaches has been to develop 'low-alcohol' yeast strains which work by redirecting their carbon metabolism away from ethanol production to other metabolites, such as glycerol. This article reviews the current challenges of producing glycerol at the expense of ethanol. It also casts new light on yeast strain development programmes which, bolstered by synthetic genomics, could potentially overcome these challenges.15 page(s