Oxidation Alters the Architecture of the Phenylalanyl-tRNA Synthetase Editing Domain to Confer Hyperaccuracy

High fidelity during protein synthesis is accomplished by aminoacyl-tRNA synthetases (aaRSs). These enzymes ligate an amino acid to a cognate tRNA and have proofreading and editing capabilities that ensure high fidelity. Phenylalanyl-tRNA synthetase (PheRS) preferentially ligates a phenylalanine to a tRNAPhe over the chemically similar tyrosine, which differs from phenylalanine by a single hydroxyl group. In bacteria that undergo exposure to oxidative stress such as Salmonella enterica serovar Typhimurium, tyrosine isomer levels increase due to phenylalanine oxidation. Several residues are oxidized in PheRS and contribute to hyperactive editing, including against mischarged Tyr-tRNAPhe, despite these oxidized residues not being directly implicated in PheRS activity. Here, we solve a 3.6 Å cryo-electron microscopy structure of oxidized S. Typhimurium PheRS. We find that oxidation results in widespread structural rearrangements in the β-subunit editing domain and enlargement of its editing domain. Oxidization also enlarges the phenylalanyl-adenylate binding pocket but to a lesser extent. Together, these changes likely explain why oxidation leads to hyperaccurate editing and decreased misincorporation of tyrosine. Taken together, these results help increase our understanding of the survival of S. Typhimurium during human infection

The tomato kinase Pti1 contributes to production of reactive oxygen species in response to two flagellin-derived peptides and promotes resistance to pseudomonas syringae Infection

The Pti1 kinase was identified from a reverse genetic screen as contributing to pattern-triggered immunity (PTI) against Pseudomonas syringae pv. tomato (Pst). The tomato genome has two Pti1 genes, referred to as Pti1a and Pti1b. A hairpin-Pti1 (hpPti1) construct was developed and was used to generate two independent stable transgenic tomato lines that had reduced transcript abundance of both genes. In response to P. syringae pv. tomato inoculation, these hpPti1 plants developed more severe disease symptoms, supported higher bacterial populations, and had reduced transcript accumulation of PTI-associated genes, as compared with wild-type plants. In response to two flagellin-derived peptides, the hpPti1 plants produced lesser amounts of reactive oxygen species (ROS) but showed no difference in mitogen-activated protein kinase (MAPK). Synthetic Pti1a and Pti1b genes designed to avoid silencing were transiently expressed in the hpPti1 plants and restored the ability of the plants to produce wild-type levels of ROS. Our results identify a new component of PTI in tomato that, because it affects ROS production but not MAPK signaling, appears to act early in the immune response.Instituto de Fisiología Vegeta

Exploring and Expanding Park Use Through Interactive Game Apps

Final project for URSP688L: Planning Technologies (Fall 2017). University of Maryland, College Park.This report compiles ways to use technology, specifically a scavenger hunt game, to bring new audiences to Montgomery County parks, in an effort to promote increased engagement and longer park visits. This project capitalizes on the popularity of the interactive game apps and uses a similar game to entice people into the parks and to expand their visits. This report is the initial data collection of a larger effort to create a scavenger hunt application (“app”) that will be developed by another class in Spring 2018. The data comes from 19 park visits and gathers data on location and features within each park. From the field survey, we gathered coordinate information and pictures of the features as well as information was used to create educational and other questions that will be part of the future game app. The fieldwork focused on specific background questions, and key features were mapped for example, the relationship to population density and the distance to park entrances. The analysis and findings resulted in several recommendations, most notably to consider multiple versions of the app based on age group and categories such as walkable urban parks or historic sites. Other recommendations include suggestions for a continual reevaluation of park amenities, increased promotion, and new ways to market the parks and create new partnerships.Montgomery Count

An investigation of Chromatium vinosum high-potential irondashsulfur protein by EPR and Mossbauer spectroscopy; evidence for a freezing-induced dimerization in NaCl solutions

The high-potential irondashsulfur protein (HiPIP) from Chromatium vinosum contains a cubane prosthetic group that shuttles between the [4Fe-4S]3+,2+ states. We find that the EPR spectra from this protein can be explained as a sum of two components, a major one with g=2.02; 2.04; 2.12, and a minor one with g=2.04; 2.07; ~2.13. In the presence of 0.1-2.0 M NaCl, freezing induces polymerization of the protein (presumably dimers), which is detected as intercluster spindashspin interaction in the EPR. The observed spindashspin interactions are interpreted as being due to two very similar dimeric structures in an approx. 1:2 ratio. Computer simulation of the X- and Q-band EPR spectra shows that the z-components of the g-tensors in each dimer pair must be co-linear, with center-to-center distances between the clusters of ~ 13 A and ~ 16 A. Inspection of possible dimeric structures of C. vinosum HiPIP by standard molecular graphics procedures revealed that the Fe/S cluster is exposed toward a flattened surface and is accessible to solvent. Moreover, the Fe/S clusters in two HiPIP molecules can easily achieve a center-to-center distance of ~ 14 A when approaching along a common 3-fold axis that extends through the S4 sulfur atom of the cubane; the z-component of the EPR g-tensor is co-linear with this symmetry axis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29129/1/0000168.pd

City of Frederick Carbon Footprint and Energy Profile

As Maryland's second largest city, it is imperative that the City of Frederick being tracking and managing its role in contributing to global greenhouse gas emissions (GHGs). This report is the City's first greenhouse gas inventory of local government operations and covers the year 2013. With the data sources, methodology, findings, and recommendations collected or generated as a part of this effort, The City of Frederick is better positioned to institutionalize the process of tracking and managing greenhouse gas emissions, energy use, and other resources that will advance sustainability.The City of Frederic

Healthy Annapolis

Final project for Urban Studies and Planning Studio (Summer 2017). University of Maryland, College Park.Annapolis, Maryland, located in Anne Arundel County, is home to the United States Naval Academy and Saint John’s College. The small waterfront capital city is also a popular tourist destination for sailors and history buffs drawn to the nationally recognized historic district. While continuing to focus on preserving the City’s historic and natural resources and strong local economy, Annapolis is taking steps to become a healthier city by participating in the Let’s Move! Cities Towns, and Counties (LMCTC) initiative, a national campaign to end childhood obesity by providing guidance to elected officials, parents, schools, community leaders, and other stakeholders in order to make healthy living accessible for everyone. Annapolis has successfully met the five initial program goals for LMCTC, and has achieved All-Star status. This report will help the City pursue three of the four All-Star strategies it is now eligible to pursue after achieving All-Star status. This report highlights disadvantaged communities, as they are more likely to suffer from poor health. In addition to an increased likelihood of health issues, these communities are also less likely to have resources such as education and community support to improve certain aspects of their health. This University of Maryland PALS summer studio project is meant to help guide the City of Annapolis in creating a healthier city for all residents, and in reaching their LMCTC All-Star strategies. Four chapters were written by groups that focused on health-related aspects of the city that relate directly to areas of focus for achieving All-Star status: 1) updates to incorporate health into the Comprehensive Plan, 2) parks and open space, 3) bicycle infrastructure, and 4) urban agriculture and community gardens. We hope that by providing recommendations for integrating health into the planning process and city design, and by suggesting strategies to make the most effective use of existing tools, Annapolis will be better situated to achieve its LMCTC All-Star strategies.The City of Annapoli

Highly pathogenic avian influenza virus infection in chickens but not ducks is associated with elevated host immune and pro-inflammatory responses

Highly pathogenic avian influenza (HPAI) H5N1 viruses cause severe infection in chickens at near complete mortality, but corresponding infection in ducks is typically mild or asymptomatic. To understand the underlying molecular differences in host response, primary chicken and duck lung cells, infected with two HPAI H5N1 viruses and a low pathogenicity avian influenza (LPAI) H2N3 virus, were subjected to RNA expression profiling. Chicken cells but not duck cells showed highly elevated immune and pro-inflammatory responses following HPAI virus infection. HPAI H5N1 virus challenge studies in chickens and ducks corroborated the in vitro findings. To try to determine the underlying mechanisms, we investigated the role of signal transducer and activator of transcription-3 (STAT-3) in mediating pro-inflammatory response to HPAIV infection in chicken and duck cells. We found that STAT-3 expression was down-regulated in chickens but was up-regulated or unaffected in ducks in vitro and in vivo following H5N1 virus infection. Low basal STAT-3 expression in chicken cells was completely inhibited by H5N1 virus infection. By contrast, constitutively active STAT-3 detected in duck cells was unaffected by H5N1 virus infection. Transient constitutively-active STAT-3 transfection in chicken cells significantly reduced pro-inflammatory response to H5N1 virus infection; on the other hand, chemical inhibition of STAT-3 activation in duck cells increased pro-inflammatory gene expression following H5N1 virus infection. Collectively, we propose that elevated pro-inflammatory response in chickens is a major pathogenicity factor of HPAI H5N1 virus infection, mediated in part by the inhibition of STAT-3

Altered Chromosomal Positioning, Compaction, and Gene Expression with a Lamin A/C Gene Mutation

Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression.To investigate the hypothesis that mutant lamin A/C changes the lamina's ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction.These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered

Large-Scale Absence of Sharks on Reefs in the Greater-Caribbean: A Footprint of Human Pressures

BACKGROUND: In recent decades, large pelagic and coastal shark populations have declined dramatically with increased fishing; however, the status of sharks in other systems such as coral reefs remains largely unassessed despite a long history of exploitation. Here we explore the contemporary distribution and sighting frequency of sharks on reefs in the greater-Caribbean and assess the possible role of human pressures on observed patterns. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed 76,340 underwater surveys carried out by trained volunteer divers between 1993 and 2008. Surveys were grouped within one km2 cells, which allowed us to determine the contemporary geographical distribution and sighting frequency of sharks. Sighting frequency was calculated as the ratio of surveys with sharks to the total number of surveys in each cell. We compared sighting frequency to the number of people in the cell vicinity and used population viability analyses to assess the effects of exploitation on population trends. Sharks, with the exception of nurse sharks occurred mainly in areas with very low human population or strong fishing regulations and marine conservation. Population viability analysis suggests that exploitation alone could explain the large-scale absence; however, this pattern is likely to be exacerbated by additional anthropogenic stressors, such as pollution and habitat degradation, that also correlate with human population. CONCLUSIONS/SIGNIFICANCE: Human pressures in coastal zones have lead to the broad-scale absence of sharks on reefs in the greater-Caribbean. Preventing further loss of sharks requires urgent management measures to curb fishing mortality and to mitigate other anthropogenic stressors to protect sites where sharks still exist. The fact that sharks still occur in some densely populated areas where strong fishing regulations are in place indicates the possibility of success and encourages the implementation of conservation measures

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead