Construction of synthetic regulatory networks in yeast

Yeast species such as Saccharomyces cerevisiae have been exploited by humans for millennia and so it is therefore unsurprising that they are attractive cells to re-engineer for industrial use. Despite many beneficial traits yeast has for synthetic biology, it currently lags behind Escherichia coli in the number of synthetic networks that have been described. While the eukaryotic nature of yeast means that its regulation is not as simple to predict as it is for E. coli, once initial considerations have been made yeast is pleasingly tractable. In this review we provide a loose guide for constructing and implementing synthetic regulatory networks in S. cerevisiae using examples from previous research to highlight available resources, specific considerations and potential future advances. © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

Urinary Acrylonitrile Metabolite Concentrations Before and after Smoked, Vaporized, and Oral Cannabis in Frequent and Occasional Cannabis Users.

Cannabis use through smoking, vaping, or ingestion is increasing, but only limited studies have investigated the resulting exposure to harmful chemicals. N-acetyl-S-(2-cyanoethyl)-L-cysteine (2CYEMA), a urinary metabolite of acrylonitrile, a possible carcinogen, is elevated in the urine of past-30-day cannabis users compared to non-cannabis users. Five frequent and five occasional cannabis users smoked and vaped cannabis on separate days; one also consumed cannabis orally. Urine samples were collected before and up to 72 h post dose and urinary 2CYEMA was quantified. We compared 2CYEMA pre-exposure levels, maximum concentration, time at maximum concentration for occasional versus frequent users following different exposure routes, and measured half-life of elimination. Smoking cannabis joints rapidly (within 10 min) increased 2CYEMA in the urine of occasional cannabis users, but not in frequent users. Urine 2CYEMA did not consistently increase following vaping or ingestion in either study group. Cigarette smokers had high pre-exposure concentrations of 2CYEMA. Following cannabis smoking, the half-lives of 2CYEMA ranged from 2.5 to 9.0 h. 2CYEMA is an effective biomarker of cannabis smoke exposure, including smoke from a single cannabis joint, however, not from vaping or when consumed orally. When using 2CYEMA to evaluate exposure in cannabis users, investigators should collect the details about tobacco smoking, route of consumption, and time since last use as possible covariates

Comparison of Trihalomethanes in Tap Water and Blood: A Case Study in the United States

Background: Epidemiological studies have used various measures to characterize trihalomethane (THM) exposures, but the relationship of these indicators to exposure biomarkers remains unclear

The importance of human dimensions research in managing harmful algal blooms

Author Posting. © Ecological Society of America, 2010. This article is posted here by permission of Ecological Society of America for personal use, not for redistribution. The definitive version was published in Frontiers in Ecology and the Environment 8 (2010): 75–83, doi:10.1890/070181.Harmful algal blooms (HABs) are natural freshwater and marine hazards that impose substantial adverse impacts on the human use of coastal and marine resources. The socioeconomic and health impacts of HABs can be considerable, thereby making a case for “human dimensions” research to support HAB response. Human dimensions research is multidisciplinary, integrating social science, humanities, and other fields with natural science to enhance resource management by addressing human causes, consequences, and responses to coastal environmental problems. Case studies reported here illustrate the importance of human dimensions research. Incorporating such research into the scientific agenda – as well as into management decisions of public agencies concerned with natural resource management, environmental protection, and public health and welfare – requires the development of both strategic guidance and institutional capacity. The recent development of a multi-agency research strategy for HAB response and a strategic plan for human dimensions research represent two important steps in this direction.This paper was developed with partial support from NOAA’s National Centers for Coastal and Ocean Science

Investigating Childhood Leukemia in Churchill County, Nevada

BACKGROUND: Sixteen children diagnosed with acute leukemia between 1997 and 2002 lived in Churchill County, Nevada, at the time of or before their illness. Considering the county population and statewide cancer rate, fewer than two cases would be expected. OBJECTIVES: In March 2001, the Centers for Disease Control and Prevention led federal, state, and local agencies in a cross-sectional, case-comparison study to determine if ongoing environmental exposures posed a health risk to residents and to compare levels of contaminants in environmental and biologic samples collected from participating families. METHODS: Surveys with more than 500 variables were administered to 205 people in 69 families. Blood, urine, and cheek cell samples were collected and analyzed for 139 chemicals, eight viral markers, and several genetic polymorphisms. Air, water, soil, and dust samples were collected from almost 80 homes to measure more than 200 chemicals. RESULTS: The scope of this cancer cluster investigation exceeded any previous study of pediatric leukemia. Nonetheless, no exposure consistent with leukemia risk was identified. Overall, tungsten and arsenic levels in urine and water samples were significantly higher than national comparison values; however, levels were similar among case and comparison groups. CONCLUSIONS: Although the cases in this cancer cluster may in fact have a common etiology, their small number and the length of time between diagnosis and our exposure assessment lessen the ability to find an association between leukemia and environmental exposures. Given the limitations of individual cancer cluster investigations, it may prove more efficient to pool laboratory and questionnaire data from similar leukemia clusters

Childhood Asthma and Environmental Exposures at Swimming Pools: State of the Science and Research Recommendations

OBJECTIVES: Recent studies have explored the potential for swimming pool disinfection by-products (DBPs), which are respiratory irritants, to cause asthma in young children. Here we describe the state of the science on methods for understanding children's exposure to DBPs and biologics at swimming pools and associations with new-onset childhood asthma and recommend a research agenda to improve our understanding of this issue. DATA SOURCES: A workshop was held in Leuven, Belgium, 21-23 August 2007, to evaluate the literature and to develop a research agenda to better understand children's exposures in the swimming pool environment and their potential associations with new-onset asthma. Participants, including clinicians, epidemiologists, exposure scientists, pool operations experts, and chemists, reviewed the literature, prepared background summaries, and held extensive discussions on the relevant published studies, knowledge of asthma characterization and exposures at swimming pools, and epidemiologic study designs. SYNTHESIS: Childhood swimming and new-onset childhood asthma have clear implications for public health. If attendance at indoor pools increases risk of childhood asthma, then concerns are warranted and action is necessary. If there is no such relationship, these concerns could unnecessarily deter children from indoor swimming and/or compromise water disinfection. CONCLUSIONS: Current evidence of an association between childhood swimming and new-onset asthma is suggestive but not conclusive. Important data gaps need to be filled, particularly in exposure assessment and characterization of asthma in the very young. Participants recommended that additional evaluations using a multidisciplinary approach are needed to determine whether a clear association exists

Synthetic yeast chromosome XI design provides a testbed for the study of extrachromosomal circular DNA dynamics

We describe construction of the synthetic yeast chromosome XI (synXI) and reveal the effects of redesign at non-coding DNA elements. The 660-kb synthetic yeast genome project (Sc2.0) chromosome was assembled from synthesized DNA fragments before CRISPR-based methods were used in a process of bug discovery, redesign, and chromosome repair, including precise compaction of 200 kb of repeat sequence. Repaired defects were related to poor centromere function and mitochondrial health and were associated with modifications to non-coding regions. As part of the Sc2.0 design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show that these sites can facilitate induced extrachromosomal circular DNA (eccDNA) formation, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI contributes to our understanding of non-coding DNA elements, provides a useful tool for eccDNA study, and will inform future synthetic genome design

Rational Diversification of a Promoter Providing Fine-Tuned Expression and Orthogonal Regulation for Synthetic Biology

Yeast is an ideal organism for the development and application of synthetic biology, yet there remain relatively few well-characterised biological parts suitable for precise engineering of this chassis. In order to address this current need, we present here a strategy that takes a single biological part, a promoter, and re-engineers it to produce a fine-graded output range promoter library and new regulated promoters desirable for orthogonal synthetic biology applications. A highly constitutive Saccharomyces cerevisiae promoter, PFY1p, was identified by bioinformatic approaches, characterised in vivo and diversified at its core sequence to create a 36-member promoter library. TetR regulation was introduced into PFY1p to create a synthetic inducible promoter (iPFY1p) that functions in an inverter device. Orthogonal and scalable regulation of synthetic promoters was then demonstrated for the first time using customisable Transcription Activator-Like Effectors (TALEs) modified and designed to act as orthogonal repressors for specific PFY1-based promoters. The ability to diversify a promoter at its core sequences and then independently target Transcription Activator-Like Orthogonal Repressors (TALORs) to virtually any of these sequences shows great promise toward the design and construction of future synthetic gene networks that encode complex “multi-wire” logic functions

Synthetic yeast chromosome XI design enables extrachromosomal circular DNA formation on demand

We describe construction of the 660 kilobase synthetic yeast chromosome XI (synXI) and reveal how synthetic redesign of non-coding DNA elements impact the cell. To aid construction from synthesized 5 to 10 kilobase DNA fragments, we implemented CRISPR-based methods for synthetic crossovers in vivo and used these methods in an extensive process of bug discovery, redesign and chromosome repair, including for the precise removal of 200 kilobases of unexpected repeated sequence. In synXI, the underlying causes of several fitness defects were identified as modifications to non-coding DNA, including defects related to centromere function and mitochondrial activity that were subsequently corrected. As part of synthetic yeast chromosome design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show here that targeted insertion of these sites can be used to create extrachromosomal circular DNA on demand, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI has uncovered effects of non-coding and extrachromosomal circular DNA, contributing to better understanding of these elements and informing future synthetic genome design

Design, Construction, and Functional Characterization of a tRNA Neochromosome in Yeast

Here we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs