Synthetic shuffling and in vitro selection reveal the rugged adaptive fitness landscape of a kinase ribozyme

The relationship between genotype and phenotype is often described as an adaptive fitness landscape. In this study, we used a combination of recombination, in vitro selection, and comparative sequence analysis to characterize the fitness landscape of a previously isolated kinase ribozyme. Point mutations present in improved variants of this ribozyme were recombined in vitro in more than 10[superscript 14] different arrangements using synthetic shuffling, and active variants were isolated by in vitro selection. Mutual information analysis of 65 recombinant ribozymes isolated in the selection revealed a rugged fitness landscape in which approximately one-third of the 91 pairs of positions analyzed showed evidence of correlation. Pairs of correlated positions overlapped to form densely connected networks, and groups of maximally connected nucleotides occurred significantly more often in these networks than they did in randomized control networks with the same number of links. The activity of the most efficient recombinant ribozyme isolated from the synthetically shuffled pool was 30-fold greater than that of any of the ribozymes used to build it, which indicates that synthetic shuffling can be a rich source of ribozyme variants with improved properties.National Institutes of Health (U.S.) (Grant GM061835

Optimal Location of the U.S. Broiler Industry

Livestock Production/Industries, Marketing, Productivity Analysis,

Searching for ribozymes with new and improved properties

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006.Includes bibliographical references.The technique of in vitro selection was used to learn more about the properties of RNA sequence space. In Chapter 1 I investigated how readily ribozymes with new catalytic activities and folds could arise from an existing ribozyme scaffold. In vitro selection was used to isolate 23 kinase ribozymes from a pool of more than 104 variants of an aminoacylase parent ribozyme. The density of kinase ribozymes in the pool increased dramatically as the mutational distance from the starting ribozyme increased, suggesting a need to escape the fold of the parent. Consistent with this idea, the folds of two kinases characterized in detail were different from that of the starting aminoacylase scaffold. In Chapter 2 I1 investigated the extent to which one of these kinase ribozymes could be optimized using a method of recombination called synthetic shuffling. Point mutations from previously isolated sequence variants of this ribozyme were shuffled in more than 1014 different combinations, and active variants isolated by in vitro selection. The rate of the most efficient ribozyme identified was 30-fold faster than that of the most efficient ribozyme used to build the pool, with a second order rate enhancement approaching 100°- fold.(cont.) Further analysis revealed two groups of mutations, derived from two different ribozymes used to build the shuffled pool, that each increased the rate of the ribozyme by approximately 30-fold. The effects of these mutations were independent of one another, and when combined produced a ribozyme with a rate 600-fold faster than that of the initial isolate. Together, these experiments provide insight into how RNA sequence space can be searched for ribozymes with new and improved properties.by Edward A. Curtis.Ph.D

Discovery of widespread GTP-binding motifs in genomic DNA and RNA

Biological RNAs that bind small-molecules have been implicated in a variety of regulatory and catalytic processes. Inspired by these examples, we used in vitro selection to search a pool of genomeencoded RNA fragments for naturally occurring GTP aptamers. Several classes of aptamers were identified, including one ("the G motif") with a G-quadruplex structure. Further analysis revealed that most RNA and DNA G-quadruplexes bind GTP. The G motif is abundant in eukaryotes, and the human genome contains ∼75,000 examples with dissociation constants comparable to the GTP concentration of a eukaryotic cell (∼300 μM). G-quadruplexes play roles in diverse cellular processes, and our findings raise the possibility that GTP may play a role in the function of these elements. Consistent with this possibility, the sequence requirements of several classes of regulatory G-quadruplexes parallel those of GTP binding.Chemistry and Chemical Biolog

The Transnational and Diasporic Future of African American Religions in the United States

This article calls forth a vision for the future study of African American religions in the United States by examining how transnational contact and diasporic consciousness have affected the past practice and are likely to affect the future practice of Christianity, Islam, and African-derived, Orisha-based religions in Black America. It offers a synthesis of scholarly literature and charts possible directions for analyzing Africana religions beyond the ideological and geographical boundaries of the nation-state. The article focuses on two primary forms of imagined and physical movement: the immigration of self-identifying Black people from Africa, Europe, the Caribbean, Mexico, and other places to the United States; and the travel, tourism, pilgrimage, and other movement—whether physical or not—of American-born Black people to places outside the Unites States

Response and Resistance to Paradox-Breaking BRAF Inhibitor in Melanomas

FDA-approved BRAF inhibitors produce high response rates and improve overall survival in patients with BRAF V600E/K-mutant melanoma, but are linked to pathologies associated with paradoxical ERK1/2 activation in wild-type BRAF cells. To overcome this limitation, a next-generation paradox-breaking RAF inhibitor (PLX8394) has been designed. Here, we show that by using a quantitative reporter assay, PLX8394 rapidly suppressed ERK1/2 reporter activity and growth of mutant BRAF melanoma xenografts. Ex vivo treatment of xenografts and use of a patient-derived explant system (PDeX) revealed that PLX8394 suppressed ERK1/2 signaling and elicited apoptosis more effectively than the FDA-approved BRAF inhibitor, vemurafenib. Furthermore, PLX8394 was efficacious against vemurafenibresistant BRAF splice variant-expressing tumors and reduced splice variant homodimerization. Importantly, PLX8394 did not induce paradoxical activation of ERK1/2 in wild-type BRAF cell lines or PDeX. Continued in vivo dosing of xenografts with PLX8394 led to the development of acquired resistance via ERK1/2 reactivation through heterogeneous mechanisms; however, resistant cells were found to have differential sensitivity to ERK1/2 inhibitor. These findings highlight the efficacy of a paradox-breaking selective BRAF inhibitor and the use of PDeX system to test the efficacy of therapeutic agents. © 2017 American Association for Cancer Research

Long-term use of antibiotics and risk of colorectal adenoma

Objective—Recent evidence suggests that antibiotic use, which alters the gut microbiome, is associated with an increased risk of colorectal cancer. However, the association between antibiotic use and risk of colorectal adenoma, the precursor for the majority of colorectal cancers, has not been investigated. Design—We prospectively evaluated the association between antibiotic use at age 20–39 and 40–59 (assessed in 2004) and recent antibiotic use (assessed in 2008) with risk of subsequent colorectal adenoma among 16,642 women aged ≥60 enrolled in the Nurses’ Health Study who underwent at least one colonoscopy through 2010. We used multivariate logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Results—We documented 1,195 cases of adenoma. Increasing duration of antibiotic use at age 20–39 (Ptrend=0.002) and 40–59 (Ptrend=0.001) was significantly associated with an increased risk of colorectal adenoma. Compared to non-users, women who used antibiotics for ≥2 months between age 20–39 had a multivariable OR of 1.36 (95% CI: 1.03–1.79). Women who used ≥2 months of antibiotics between age 40–59 had a multivariable OR of 1.69 (95% CI: 1.24–2.31). The associations were similar for low-risk vs. high-risk adenomas (size ≥1 cm, or with tubulovillous/villous histology, or ≥3 detected lesions), but appeared modestly stronger for proximal compared with distal adenomas. In contrast, recent antibiotic use within the past 4 years was not associated with risk of adenoma (Ptrend=0.44). Conclusions—Long-term antibiotic use in early to middle adulthood was associated with increased risk of colorectal adenoma

Computational Fluid Dynamics Uncertainty Analysis Applied to Heat Transfer over a Flat Plate

There have been few discussions on using Computational Fluid Dynamics (CFD) without experimental validation. Pairing experimental data, uncertainty analysis, and analytical predictions provides a comprehensive approach to verification and is the current state of the art. With pressed budgets, collecting experimental data is rare or non-existent. This paper investigates and proposes a method to perform CFD uncertainty analysis only from computational data. The method uses current CFD uncertainty techniques coupled with the Student-T distribution to predict the heat transfer coefficient over a at plate. The inputs to the CFD model are varied from a specified tolerance or bias error and the difference in the results are used to estimate the uncertainty. The variation in each input is ranked from least to greatest to determine the order of importance. The results are compared to heat transfer correlations and conclusions drawn about the feasibility of using CFD without experimental data. The results provide a tactic to analytically estimate the uncertainty in a CFD model when experimental data is unavailabl

Multispecies diel transcriptional oscillations in open ocean heterotrophic bacterial assemblages

Oscillating diurnal rhythms of gene transcription, metabolic activity, and behavior are found in all three domains of life. However, diel cycles in naturally occurring heterotrophic bacteria and archaea have rarely been observed. Here, we report time-resolved whole-genome transcriptome profiles of multiple, naturally occurring oceanic bacterial populations sampled in situ over 3 days. As anticipated, the cyanobacterial transcriptome exhibited pronounced diel periodicity. Unexpectedly, several different heterotrophic bacterioplankton groups also displayed diel cycling in many of their gene transcripts. Furthermore, diel oscillations in different heterotrophic bacterial groups suggested population-specific timing of peak transcript expression in a variety of metabolic gene suites. These staggered multispecies waves of diel gene transcription may influence both the tempo and the mode of matter and energy transformation in the sea.Gordon and Betty Moore Foundation (GBMF 492.01)Gordon and Betty Moore Foundation (GBMF 3777)National Science Foundation (U.S.) (Grant EF0424599)David & Lucile Packard Foundatio

Atomic oxygen durability evaluation of the flexible batten for the photovoltaic array mast on Space Station

A test program was conducted at the National Aeronautics and Space Administration's Lewis Research Center (LeRC) to evaluate the long term low Earth orbital (LEO) atomic oxygen (AO) durability of a flexible (fiberglass-epoxy composite) batten. The flexible batten is a component used to provide structural rigidity in the photovoltaic array mast on Space Station. The mast is used to support and articulate the photovoltaic array, therefore, the flexible batten must be preloaded for the 15 year lifetime of an array blanket. Development hardware and composite materials were evaluated in ground testing facilities for AO durability and dynamic retraction-deployment cyclic loading representative of expected full life in-space application. The CV1144 silicone (AO protective) coating was determined to provide adequate protection against AO degradation of the composite material and provided fiber containment, thus the structural integrity of the flexible batten was maintained. Both silicone coated and uncoated flexible battens maintained load carrying capabilities. Results of the testing did indicate that the CV1144 silicone protective coating was oxidized by AO reactions to form a brittle glassy (SiO2) skin that formed cracking patterns on all sides of the coated samples. The cracking was observed in samples that were mechanically stressed as well as samples in non-stressed conditions. The oxidized silicon was observed to randomly spall in small localized areas, on the flexible battens that underwent retraction-deployment cycling. Some darkening of the silicon, attributed to vacuum ultraviolet (VUV) radiation, was observed