Transcending the prediction paradigm: novel applications of SHAPE to RNA function and evolution

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) provides information on RNA structure at single-nucleotide resolution. It is most often used in conjunction with RNA secondary structure prediction algorithms as a probabilistic or thermodynamic restraint. With the recent advent of ultra-high-throughput approaches for collecting SHAPE data, the applications of this technology are extending beyond structure prediction. In this review, we discuss recent applications of SHAPE data in the transcriptomic context and how this new experimental paradigm is changing our understanding of these experiments and RNA folding in general. SHAPE experiments probe both the secondary and tertiary structure of an RNA, suggesting that model-free approaches for within and comparative RNA structure analysis can provide significant structural insight without the need for a full structural model. New methods incorporating SHAPE at different nucleotide resolutions are required to parse these transcriptomic data sets to transcend secondary structure modeling with global structural metrics. These 'multiscale' approaches provide deeper insights into RNA global structure, evolution, and function in the cell. For further resources related to this article, please visit the WIREs website

From Neurons to Nucleic Acids: Spatio-temporal Emergent Behaviors of Complex Biological Systems

Biological systems, from the molecular to the organismal level, demonstrate emergent behaviors that form fundamental characteristics of the system. Many biological phenomena are difficult to observe experimentally because of technical limitations. Computational models are a useful tool for interpretation of behaviors of complex biological systems. This dissertation examines models for two different types of emergent behaviors: cortical state and RNA structure. In Chapter 2, I use a computational neural model to understand the effects of neurons with long-range projections and propagation delays. I find that propagation delays cause a local network to exhibit a variety of metastable network states. Application of transcranial alternating current stimulation enables the switching of a network to a different metastable state. These emergent behaviors of a network of modeled neurons are a simplified version of neocortical states, and the results provide a foundation for future research on the effects of stimulation on cortical behavior. In Chapter 3, I examine the structure of the 5′ UTR of the human tumor suppressor gene RB1 using an experimentally-directed RNA structural model. The 5′ UTR adopts three distinct structures with similar frequencies. Two disease-associated mutations each collapse the structural ensemble into a single structure, and also affect translation efficiency. By creating structural models of two homologous UTRs, I find that the ability to adopt multiple conformations is a conserved feature of this UTR and that RNA structure regulates this transcript. In Chapter 4, I model RNA structure in Sindbis virus (SINV). SINV is a single-stranded RNA virus, with known functional elements within its RNA genome. I created experimentally-directed structural models for highly structured portions of the genome. By disrupting these structures through systematic mutational design, I identified regulatory RNA elements within the genome. Most structures within the genome are not conserved in related species of virus, indicating that this virus is highly structurally divergent and utilizes its evolutionary space to create new structures. These three projects present three different ways of using computational models to characterize complex biological systems. Informed by biological data, computational models provide further insight into the role of these emergent behaviors within a system.Doctor of Philosoph

Sensorimotor Behavioral Tests for Use in a Juvenile Rat Model of Traumatic Brain Injury: Assessment of Sex Differences

Modeling juvenile traumatic brain injury (TBI) in rodents presents several unique challenges compared to adult TBI, one of which is selecting appropriate sensorimotor behavioral tasks that enable the assessment of the extent of injury and recovery over time in developing animals. To address this challenge, we performed a comparison of common sensorimotor tests in Long-Evans rats of various sizes and developmental stages (postnatal days 16–45, 35–190 g). Tests were compared and selected for their developmental appropriateness, scalability for growth, pre-training requirements, and throughput capability. Sex differences in response to TBI were also assessed. Grid walk, automated gait analysis, rotarod, beam walk, spontaneous forelimb elevation test, and measurement of motor activity using the force-plate actometer were evaluated. Grid walk, gait analysis, and rotarod failed to meet one or more of the evaluation criteria. Beam walk, spontaneous forelimb elevation test, and measurement of motor activity using the force-plate actometer satisfied all criteria and were capable of detecting motor abnormalities in rats subjected to controlled cortical impact on postnatal day 17. No sex differences were detected in the acute effects of TBI or functional recovery during the 28 days after injury using these tests. This demonstrates the utility of these tests for the evaluation of sensorimotor function in studies using rat models of pediatric TBI, and suggest that pre-pubertal males and females respond similarly to TBI with respect to sensorimotor outcomes

A 2000 year varve-based climate record from the central Brooks Range, Alaska

Varved minerogenic sediments from glacial-fed Blue Lake, northern Alaska, are used to investigate late Holocene climate variability. Varve-thickness measurements track summer temperature recorded at Atigun Pass, located 41 km east at a similar elevation (r2 = 0.31, P = 0.08). Results indicate that climate in the Brooks Range from 10 to 730 AD (varve year) was warm with precipitation inferred to be higher than during the twentieth century. The varve-temperature relationship for this period was likely compromised and not used in our temperature reconstruction because the glacier was greatly reduced, or absent, exposing sub-glacial sediments to erosion from enhanced precipitation. Varve-inferred summer temperatures and precipitation decreased after 730 AD, averaging 0.4°C above the last millennial average (LMA = 4.2°C) from 730 to 850 AD, and 0.1°C above the LMA from 850 to 980 AD. Cooling culminated between 980 and 1030 AD with temperatures 0.7°C below the LMA. Varve-inferred summer temperatures increased between 1030 and 1620 AD to the LMA, though the period between 1260 and 1350 AD was 0.2°C below the LMA. Although there is no equivalent to the European Medieval Warm Period in the Blue Lake record, two warm intervals occurred from 1350 to 1450 AD and 1500 to 1620 AD (0.4 and 0.3°C above the LMA, respectively). During the Little Ice Age (LIA; 1620 to 1880 AD), inferred summer temperature averaged 0.2°C below the LMA. After 1880 AD, inferred summer temperature increased to 0.8°C above the LMA, glaciers retreated, but aridity persisted based on a number of regional paleoclimate records. Despite warming and glacial retreat, varve thicknesses have not achieved pre-730 AD levels. This reflects limited sediment availability and transport due to a less extensive retreat compared to the first millennium, and continued relative aridity. Overall, the Blue Lake record is similar to varve records from the eastern Canadian Arctic that document a cool LIA and twentieth century warming. However, the occurrence and timing of events, such as the LIA and Medieval Warm Period, varies considerably among records, suggesting heterogeneous climatic patterns across the North American Arctic

Synthesis of zeolites from coal fly ash using mine waters

In this study mine waters obtained from coal mining operations in South Africa were used as a substitute for pure water during the synthesis of zeolites from South African coal fly ash. Procedures that had been optimized to produce single phase zeolite Na-P1 and X using pure water were employed independently. The use of circumneutral mine water resulted in similar quality zeolite Na-P1 and X whereas the use of acidic mine drainage led to the formation of a single phase hydroxysodalite zeolite. Since these two wastes (fly ash and mine waters) are found in close proximity to each other, this study demonstrates that they can be used to ameliorate each other and at the same time produce saleable zeolitic products that can be used to offset their costs of disposal and treatment.Web of Scienc

Structural divergence creates new functional features in alphavirus genomes

Alphaviruses are mosquito-borne pathogens that cause human diseases ranging from debilitating arthritis to lethal encephalitis. Studies with Sindbis virus (SINV), which causes fever, rash, and arthralgia in humans, and Venezuelan equine encephalitis virus (VEEV), which causes encephalitis, have identified RNA structural elements that play key roles in replication and pathogenesis. However, a complete genomic structural profile has not been established for these viruses. We used the structural probing technique SHAPE-MaP to identify structured elements within the SINV and VEEV genomes. Our SHAPE-directed structural models recapitulate known RNA structures, while also identifying novel structural elements, including a new functional element in the nsP1 region of SINV whose disruption causes a defect in infectivity. Although RNA structural elements are important for multiple aspects of alphavirus biology, we found the majority of RNA structures were not conserved between SINV and VEEV. Our data suggest that alphavirus RNA genomes are highly divergent structurally despite similar genomic architecture and sequence conservation; still, RNA structural elements are critical to the viral life cycle. These findings reframe traditional assumptions about RNA structure and evolution: rather than structures being conserved, alphaviruses frequently evolve new structures that may shape interactions with host immune systems or co-evolve with viral proteins

Chemical and Physical Characterization of Fly Ash as Geopolymer Material

Research on finding suitable cement substitute material becomes massive due to environmental effect. Geopolymer as inorganic material is potential to be the smart solution to overcome global warming issue. Fly ash is a waste material rich in silica and alumina becomes popular raw material to produce geopolymer. The best properties ofgeopolymer paste come from the high quality of fly ash. Therefore, it is important to investigate various types of fly ash and geopolymer properties. Their chemical and physical properties characterized by XRF, pH value, XRD and SEM. The results showed that type of fly ash depended on amount of Si-based of Ca-based compound which consisted of spherical morphology. Geopolymer paste produced from the ash with different compound has bulky and irregular shape morphology. The pH value of each ash has also a correlation with the setting time of fresh paste

A functional riboSNitch in the 3' untranslated region of FKBP5 alters MicroRNA-320a binding efficiency and mediates vulnerability to chronic post-traumatic pain

Previous studies have shown that common variants of the gene coding for FK506-binding protein 51 (FKBP5), a critical regulator of glucocorticoid sensitivity, affect vulnerability to stress-related disorders. In a previous report, FKBP5 rs1360780 was identified as a functional variant because of its effect on gene methylation. Here we report evidence for a novel functional FKBP5 allele, rs3800373. This study assessed the association between rs3800373 and post-traumatic chronic pain in 1607 women and men from two ethnically diverse human cohorts. The molecular mechanism through which rs3800373 affects adverse outcomes was established via in silico, in vivo, and in vitro analyses. The rs3800373 minor allele predicted worse adverse outcomes after trauma exposure, such that individuals with the minor (risk) allele developed more severe post-traumatic chronic musculoskeletal pain. Among these individuals, peritraumatic circulating FKBP5 expression levels increased as cortisol and glucocorticoid receptor (NR3C1) mRNA levels increased, consistent with increased glucocorticoid resistance. Bioinformatic, in vitro, and mutational analyses indicate that the rs3800373 minor allele reduces the binding of a stress-and pain-associated microRNA, miR-320a, to FKBP5 via altering the FKBP5 mRNA 3'UTR secondary structure (i.e., is a riboSNitch). This results in relatively greater FKBP5 translation, unchecked by miR-320a. Overall, these results identify an important gene–miRNA interaction influencing chronic pain risk in vulnerable individuals and suggest that exogenous methods to achieve targeted reduction in poststress FKBP5 mRNA expression may constitute useful therapeutic strategies

Multi-scale x-ray computed tomography analysis of coal microstructure and permeability changes as a function of effective stress

Gas permeability (k) and porosity (f) are the most important parameters in CBM/ECBM and CCS in deep unmineable coal seams. k and f depend on the coal microstructure, and k and f significantly change with varying effective stress. However, how the coal microstructure is related to such permeability and porosity changes is only poorly understood. We thus imaged sub-bituminous coal samples at two resolutions (medium - 33.7 µm and high - 3.43 µm voxel size) in 3D with an x-ray micro-computed tomography as a function of applied effective stress; and investigated how cleat morphology, k and f are influenced by the changes in effective stress and how these parameters are interrelated. In the images, three phases were identified: microcleats (void), a mineral phase (carbonate) and the coal matrix. When effective stress increased, the cleats became narrow and closed or disconnected. This resulted in a dramatic permeability drop with increasing effective stress, while porosity decreased only linearly

Swelling effect on coal micro structure and associated permeability reduction

© 2016 Elsevier Ltd. All rights reserved.Porosity and permeability of deep unmineable coal seams are key parameters in the context of (enhanced) coalbed methane recovery and CO2 geo-storage in coal beds as they determine productivity and injection rate. Porosity and permeability are again determined by the micro-structure of the coal, and the cleat network-coal matrix system. Furthermore, it is well established that swelling of the coal matrix due to water adsorption can significantly reduce permeability. However, the exact effect of swelling due to water adsorption on the coal micro-structure is only poorly understood, and how this microstructural change impacts on the permeability and porosity characteristics of the coal. We thus imaged dry coal plugs and swollen coal plugs (swollen due to brine adsorption) at high resolution (3.43 µm3) in 3D with an X-ray micro-computed tomograph (microCT). On the microCT images two types of cleats were identified; cleats in the coal matrix and cleats syngeneic with the mineral phase. Approximately 80% of the coal matrix cleats closed upon water adsorption, while the cleats in the mineral phase were not affected. This cleat closure by water adsorption dramatically reduced porosity and particularly permeability, consistent with dynamic permeability core-flood measurements