Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles.

The direct genetically encoded cell-based synthesis of non-natural peptide and depsipeptide macrocycles is an outstanding challenge. Here we programme the encoded synthesis of 25 diverse non-natural macrocyclic peptides, each containing two non-canonical amino acids, in Syn61Δ3-derived cells; these cells contain a synthetic Escherichia coli genome in which the annotated occurrences of two sense codons and a stop codon, and the cognate transfer RNAs (tRNAs) and release factor that normally decode these codons, have been removed. We further demonstrate that pyrrolysyl-tRNA synthetase/tRNA pairs from distinct classes can be engineered to direct the co-translational incorporation of diverse alpha hydroxy acids, with both aliphatic and aromatic side chains.

Sense codon reassignment enables viral resistance and encoded polymer synthesis.

It is widely hypothesized that removing cellular transfer RNAs (tRNAs)-making their cognate codons unreadable-might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in , we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses.

Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS.

We previously developed REXER (Replicon EXcision Enhanced Recombination); this method enables the replacement of >100 kb of the Escherichia coli genome with synthetic DNA in a single step and allows the rapid identification of non-viable or otherwise problematic sequences with nucleotide resolution. Iterative repetition of REXER (GENESIS, GENomE Stepwise Interchange Synthesis) enables stepwise replacement of longer contiguous sections of genomic DNA with synthetic DNA, and even the replacement of the entire E. coli genome with synthetic DNA.

Reprogramming the genetic code.

The encoded biosynthesis of proteins provides the ultimate paradigm for high-fidelity synthesis of long polymers of defined sequence and composition, but it is limited to polymerizing the canonical amino acids. Recent advances have built on genetic code expansion - which commonly permits the cellular incorporation of one type of non-canonical amino acid into a protein - to enable the encoded incorporation of several distinct non-canonical amino acids. Developments include strategies to read quadruplet codons, use non-natural DNA base pairs, synthesize completely recoded genomes and create orthogonal translational components with reprogrammed specificities.

Programmed chromosome fission and fusion enable precise large-scale genome rearrangement and assembly.

The design and creation of synthetic genomes provide a powerful approach to understanding and engineering biology. However, it is often limited by the paucity of methods for precise genome manipulation. Here, we demonstrate the programmed fission of the genome into diverse pairs of synthetic chromosomes and the programmed fusion of synthetic chromosomes to generate genomes with user-defined inversions and translocations.

Total synthesis of Escherichia coli with a recoded genome.

Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms.

[Positioning Ecuador in the global health agenda as a result of sector reform].

Objective: Analyze strategies implemented by Ecuador's Ministry of Public Health (MPH) to position the country in the global health agenda during the period 2011-2015 as a result of health sector reform.

Method: Documentary review and interviews with stakeholders in national and international agencies with respect to positioning in the global health sphere during the study period.

Results: It was observed that the reform process produced a new framework to manage international health relations.

[Human resources for health in Ecuador's new model of care].

Objective: Describe strategies implemented by Ecuador's Ministry of Public Health (MPH) to strengthen human resources for health leadership and respond to the new model of care, as a part of the reform process in the period 2012-2015.

Methods: A documentary review was carried out of primary and secondary sources on development of human resources for health before and after the reform.

Results: In the study period, Ecuador developed a new institutional and regulatory framework for developing human resources for health to respond to the requirements of a model of care based on primary health care.

Relative yield loss calculations in wheat (Triticum durum Desf. cv. Camacho) due to ozone exposure.

In this work, an estimation of the relative yield losses of wheat due to ozone exposure is made by means of two approaches proposed by the CLRTAP (Convention on Long Range Transboundary Air Pollution): the exposure-response approach, which deals with the exposure of plants to ozone during a certain time, and the accumulated uptake approach, which, besides ozone exposure, deals with the velocity of absorption of the contaminant and the environmental factors that modulate that absorption. Once the relative yield losses are calculated by means of the two approaches, the aim is to establish which index (the exposure-response index or the accumulated uptake index) best characterizes the response of wheat plants to ozone. The relative yield losses are compared considering two watering regimes: well watered and nonwatered.

Quantification of mesophyll resistance and apoplastic ascorbic acid as an antioxidant for tropospheric ozone in durum wheat (Triticum durum Desf. cv. Camacho).

The daily variations in cellular and apoplastic ascorbic acid and dehydroascorbic acid levels in a Mediterranean durum wheat cultivar (Triticum durum Desf. cv. Camacho) were analyzed in order to relate them to ambient ozone exposure and to subsequent stomatally absorbed ozone fluxes.

Soil water availability modulation over estimated relative yield losses in wheat (Triticum aestivum L.) due to ozone exposure.

The approach developed by Fuhrer in 1995 to estimate wheat yield losses induced by ozone and modulated by the soil water content (SWC) was applied to the data on Catalonian wheat yields. The aim of our work was to apply this approach and adjust it to Mediterranean environmental conditions by means of the necessary corrections. The main objective pursued was to prove the importance of soil water availability in the estimation of relative wheat yield losses as a factor that modifies the effects of tropospheric ozone on wheat, and to develop the algorithms required for the estimation of relative yield losses, adapted to the Mediterranean environmental conditions.

Soil CO2 efflux in a mixed pine-oak forest in Valsaín (central Spain).

Soil-surface CO2 efflux and its spatial and temporal variation were investigated in a southern Mediterranean, mixed pine-oak forest ecosystem on the northern slopes of the Sierra de Guadarrama in Spain from February 2006 to July 2006. Measurements of soil CO2 efflux, soil temperatures, and moisture were conducted in nine 1963-m2 sampling plots distributed in a gradient around the ecotone between Pinus sylvestris L. and Quercus pyrenaica Lam.