Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds.

Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.

The development of folate-functionalised palladium nanoparticles for folate receptor targeting in breast cancer cells.

Folate receptor-targeted therapy has excellent prospects for the treatment of breast cancer. A non-toxic concentration of folate-conjugated palladium-based nanoparticles was used to target the overexpressed folate receptor on breast cancer cells. The folate-conjugated nanoparticles were tailored to accumulate selectively in cancer cells relative to normal cells via the folate receptor. The MDA-MB-231, MDA-MB-468, MCF-7 breast cancer cell lines, and MCF-10A normal cell lines were used in the study. Qualitative and quantitative analysis of nanoparticle cellular uptake and accumulation was conducted using transmission electron microscopy and inductively coupled plasma-optical emission spectroscopy. The findings proved that folate-conjugated palladium nanoparticles successfully and preferentially accumulated in breast cancer cells. We conclude that folate-conjugated palladium nanoparticles can be potentially used to target breast cancer cells for radiopharmaceutical applications.

Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay.

Engineered metabolic pathways often suffer from flux imbalances that can overburden the cell and accumulate intermediate metabolites, resulting in reduced product titers. One way to alleviate such imbalances is to adjust the expression levels of the constituent enzymes using a combinatorial expression library. Typically, this approach requires high-throughput assays, which are unfortunately unavailable for the vast majority of desirable target compounds. To address this, we applied regression modeling to enable expression optimization using only a small number of measurements. We characterized a set of constitutive promoters in Saccharomyces cerevisiae that spanned a wide range of expression and maintained their relative strengths irrespective of the coding sequence. We used a standardized assembly strategy to construct a combinatorial library and express for the first time in yeast the five-enzyme violacein biosynthetic pathway. We trained a regression model on a random sample comprising 3% of the total library, and then used that model to predict genotypes that would preferentially produce each of the products in this highly branched pathway. This generalizable method should prove useful in engineering new pathways for the sustainable production of small molecules.

Employing a combinatorial expression approach to characterize xylose utilization in Saccharomyces cerevisiae.

Fermentation of xylose, a major constituent of lignocellulose, will be important for expanding sustainable biofuel production. We sought to better understand the effects of intrinsic (genotypic) and extrinsic (growth conditions) variables on optimal gene expression of the Scheffersomyces stipitis xylose utilization pathway in Saccharomyces cerevisiae by using a set of five promoters to simultaneously regulate each gene. Three-gene (xylose reductase, xylitol dehydrogenase (XDH), and xylulokinase) and eight-gene (expanded with non-oxidative pentose phosphate pathway enzymes and pyruvate kinase) promoter libraries were enriched under aerobic and anaerobic conditions or with a mutant XDH with altered cofactor usage. Through characterization of enriched strains, we observed (1) differences in promoter enrichment for the three-gene library depending on whether the pentose phosphate pathway genes were included during the aerobic enrichment; (2) the importance of selection conditions, where some aerobically-enriched strains underperform in anaerobic conditions compared to anaerobically-enriched strains; (3) improved growth rather than improved fermentation product yields for optimized strains carrying the mutant XDH compared to the wild-type XDH.

Pairwise selection assembly for sequence-independent construction of long-length DNA.

The engineering of biological components has been facilitated by de novo synthesis of gene-length DNA. Biological engineering at the level of pathways and genomes, however, requires a scalable and cost-effective assembly of DNA molecules that are longer than approximately 10 kb, and this remains a challenge. Here we present the development of pairwise selection assembly (PSA), a process that involves hierarchical construction of long-length DNA through the use of a standard set of components and operations. In PSA, activation tags at the termini of assembly sub-fragments are reused throughout the assembly process to activate vector-encoded selectable markers. Marker activation enables stringent selection for a correctly assembled product in vivo, often obviating the need for clonal isolation. Importantly, construction via PSA is sequence-independent, and does not require primary sequence modification (e.g. the addition or removal of restriction sites). The utility of PSA is demonstrated in the construction of a completely synthetic 91-kb chromosome arm from Saccharomyces cerevisiae.

Personality traits, perceived risk, and risk-reduction behaviors: a further study of smoking and radon.

Personality traits and risk perceptions were examined as predictors of changes in smoking behavior. Participants (N = 697) were part of a randomized controlled trial of interventions to reduce exposure to the combined hazard of radon and cigarette smoke. Participants with higher perceived risk at baseline for the combination of smoking and radon were more likely to have a more restrictive household smoking ban in place at 12-month follow-up (p < .05). Risk perceptions also predicted reductions in the total number of cigarettes smoked in the home for participants in the video intervention who had high or moderate levels of extraversion (p < .01). Greater perceived risk predicted whether highly or moderately conscientious women quit smoking (p < .05). The moderating effects of personality traits should be considered when evaluating risk-reduction interventions.

Model for the breakup of a tuft of fibers.

A simple model for the forces acting on a single fiber as it is withdrawn from a tangled fiber assembly is proposed. Particular emphasis is placed on understanding the dynamics of the reptating fiber with respect to the entanglement of fibers within the tuft. The resulting two-parameter model captures the qualitative features of experimental simulation. The model is extended to describe the breakup of a tuft. The results show good agreement with experiment and indicate where a tuft is most likely to fracture based on the density of fiber endpoints.

A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly.

Saccharomyces cerevisiae is an increasingly attractive host for synthetic biology because of its long history in industrial fermentations. However, until recently, most synthetic biology systems have focused on bacteria. While there is a wealth of resources and literature about the biology of yeast, it can be daunting to navigate and extract the tools needed for engineering applications. Here we present a versatile engineering platform for yeast, which contains both a rapid, modular assembly method and a basic set of characterized parts. This platform provides a framework in which to create new designs, as well as data on promoters, terminators, degradation tags, and copy number to inform those designs. Additionally, we describe genome-editing tools for making modifications directly to the yeast chromosomes, which we find preferable to plasmids due to reduced variability in expression. With this toolkit, we strive to simplify the process of engineering yeast by standardizing the physical manipulations and suggesting best practices that together will enable more straightforward translation of materials and data from one group to another. Additionally, by relieving researchers of the burden of technical details, they can focus on higher-level aspects of experimental design.

70 Gy thoracic radiotherapy is feasible concurrent with chemotherapy for limited-stage small-cell lung cancer: analysis of Cancer and Leukemia Group B study 39808.

PURPOSE: To prospectively evaluate the feasibility of delivering 70 Gy once-daily thoracic radiotherapy (TRT), concurrent with chemotherapy, in the treatment of limited-stage small-cell lung cancer (L-SCLC). MATERIALS AND METHODS: Eligible patients received two cycles of induction paclitaxel (175 mg/m(2) on Day 1) and topotecan (1 mg/m(2) on Days 1-5) with granulocyte colony stimulating factor support, followed by three cycles of carboplatin (area under the curve = 5 on Day 1) and etoposide (100 mg/m(2) on Days 1-3). TRT (70 Gy, 2 Gy/fx/7 weeks) was initiated with the first cycle of carboplatin and etoposide. Prophylactic cranial irradiation was offered to patients achieving a complete response or good partial response. RESULTS: Ninety percent of patients (57 of 63) proceeded to protocol TRT. There was one treatment-related fatality. Nonhematologic Grade 3/4 toxicities affecting more than 10% of patients, during or after TRT, were dysphagia (16%/5%) and febrile neutropenia (12%/4%). The response rate to all therapy was 92% and the median overall survival is 22.4 months (95% confidence interval 16.1, infinity ). Twenty-eight patients remain alive with a median follow-up of 24.7 months. CONCLUSION: 70 Gy once-daily TRT can be delivered safely in the cooperative group setting for patients with L-SCLC. Initial efficacy data are encouraging. The hypothesis that high-dose once-daily TRT results in comparable or improved survival compared with twice-daily accelerated TRT warrants testing in a Phase III trial.

Selection of chromosomal DNA libraries using a multiplex CRISPR system.

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.

Using radon risk to motivate smoking reduction II: randomized evaluation of brief telephone counseling and a targeted video.

Radon and cigarette smoking have synergistic effects on lung cancer risk. Electric utility company bill stuffers offered free radon test kits to households with at least one smoker. Participating households (n = 1364) were randomized within a 2 x 2 design to evaluate the main effects of brief telephone counseling and a targeted video on smoking cessation and the establishment of new household smoking bans. Phone counseling was associated with cessation at 3-month follow-up but neither intervention led to 12-month or sustained cessation. While neither intervention had a significant effect on new bans, there were trends in the predicted direction and the combination of the two significantly increased new bans compared with no intervention. The presence of children in the household was associated with new bans. While few households had high levels of radon, such levels were associated with radon mitigation behaviors. Together with a previous study, these results suggest radon risk is a useful and inexpensive way to engage smoking households in risk reduction behaviors, especially the institution of household smoking bans.

Measurements and modeling of the volume scattering function in the coastal northern Adriatic Sea.

We performed measurements of the volume scattering function (VSF) between 0.5 degrees and 179 degrees with an angular resolution of 0.3 degrees in the northern Adriatic Sea onboard an oceanographic platform during three different seasons, using the multispectral volume scattering meter (MVSM) instrument. We observed important differences with respect to Petzold's commonly used functions, whereas the Fournier-Forand's analytical formulation provided a rather good description of the measured VSF. The comparison of the derived scattering, b(p)(lambda) and backscattering, b(bp)(lambda) coefficients for particles with the measurements performed with the classical AC-9 and Hydroscat-6 showed agreement to within 20%. The use of an empirical relationship for the derivation of b(b)(lambda) from beta(psi,lambda) at psi=140 degrees was validated for this coastal site although psi=118 degrees was confirmed to be the most appropriate angle. The low value of the factor used to convert beta(psi,lambda) into b(b)(lambda) within the Hydroscat-6 processing partially contributed to the underestimation of b(b)(lambda) with respect to the MVSM. Finally, use of the Kopelevich model together with a measurement of b(p)(lambda) at lambda=555 nm allowed us to reconstruct the VSF with average rms percent differences between 8 and 15%.

Spectral variation of the volume scattering function measured over the full range of scattering angles in a coastal environment.

The spectral volume scattering function (VSF) was measured in a coastal environment from 0.6 degrees to 177.3 degrees by use of a recently developed device. The spectral variations of the particulate VSF and phase function (i.e., ratio of the VSF to the scattering coefficient) were examined as a function of the scattering angle. The angular dependency of both VSF and phase- function spectra was highly sensitive to the absorption and to the size distribution of the particles. As a result, the use of spectrally neutral phase functions in radiative-transfer modeling is questioned.

Assessing perceptions of synergistic health risk: a comparison of two scales.

Two approaches to measuring perceptions of synergistic risk were compared, one using the traditional Likert scale, the other using an anchored, relative scale. Perception of synergistic risk was defined as rating the combined hazard as more risky than each of its constituent single hazards. In a within-subjects design, a convenience sample from the community (N= 604) rated three hazard combinations and their constituents: Driving while Intoxicated (familiar, high synergy), Radon and Smoking (unfamiliar, high synergy), and Smoking and Driving (familiar, low synergy), on both scales. The relative scale was expected to be a more sensitive measure of synergy than the Likert scale. The effects of item order (single hazards rated first versus combined hazards rated first) were examined between subjects. Driving while Intoxicated was perceived by the large majority of participants as a synergistic risk on both scales, but neither of the other two combined hazards were rated synergistically on either scale. The relative scale demonstrated a slight advantage over the Likert scale, and presenting the single hazards first for the relative scale produced more synergistic ratings. It is recommended that anchored, relative scales be used to measure synergy and that single hazards be presented prior to the combined hazards when using relative scales.

Psychotropic medications prescribing patterns for children and adolescents in New York's public mental health system.

CONTEXT: Breakthroughs in the development of effective medications for a number of psychiatric disorders have led to increased use of these compounds in the treatment of children. OBJECTIVES: To understand the use of psychotropic medications in the treatment of children, a state-wide study was undertaken based on the data collected in a large planning study. DATA AND SETTING: A stratified random sample of 10 different program types in New York State produced data on children served in different specialty mental health services. PARTICIPANTS: Randomly selected cases were reviewed at a randomly selected sites to generate a sample of 1592 cases on which data were collected on clinical presentation and service use, including psychotropic medication prescriptions. MAIN OUTCOME MEASURES: The Child and Adolescent Needs and Strengths (CANS-MH) tool was used to provide a reliable review of clinical indicators. RESULTS: Psychotropic medication use is common in the children's public mental health service system in New York. Most children served in high intensity settings receive medication as a part of their treatment. It appears that most prescriptions for stimulants and antidepressants are consistent with either diagnostic or symptom indications. Many children with these indications are not on medications. On the other hand, a large number of children without evidence of psychosis receive antipsychotic medications. CONCLUSION: The evidence suggests that stimulant and antidepressant are not over-prescribed. However, the use of antipsychotic medications for other indications is a priority for further research.