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Despite the rapid development of novel THz sources in the last few decades, free electron lasers remain attractive due to their unique advantages including frequency tunability and high peak power. While most THz-FEL facilities use optical cavities to build power over many passes, the ability to extract a significant fraction of the beam energy in a single pass paves the way towards high average power as the repetition rate of electron sources is increased. In this dissertation, we study theoretically and experimentally a compact THz-FEL for improved single pass efficiency. At long wavelengths, FEL gain is limited by diffraction as well as slippage between the radiation and electron beam, requiring long bunch lengths for a sustained interaction. The introduction of a waveguide transversely confines the radiation and can be chosen to match the subluminal group velocity to the longitudinal electron beam velocity. This so-called zero-slippage operation extends the interaction length for short, high current beams allowing us to leverage developments in high brightness RF photoinjector sources. Strong seeding with a prebunched beam enables large decelerating gradients where resonance is maintained with strong undulator tapering, enhancing the extraction efficiency. After beginning with the theory of waveguide FELs and zero-slippage resonance, we present our simulation code GPTFEL, a custom element built on top of the 3D particle tracking code General Particle Tracer. The code simulates free-space and waveguide FEL interactions by decomposing the electromagnetic fields into a basis of frequency and spatial modes, evolving the complex amplitudes according to energy conservation with the beam and enabling start-to-end beamline simulations within a single code. Waveguide dispersion is naturally included and for free-space interactions, a source dependent expansion is implemented to limit the required number of transverse modes.Two Tessatron experiments were performed on the

Adenosine-to-inosine RNA editing catalyzed by the family of enzymes Adenosine Deaminases Acting on RNA (ADARs) plays several important roles in biological homeostasis of metazoans. Hydrolytic deamination of adenosine by ADARs results in the formation of inosine, which is read by cellular machinery as guanosine. This A-to-G information change can impact miRNA/siRNA biogenesis, splicing, mRNA recoding, and the innate immune response. Humans encode three ADAR genes, with ADAR1 and ADAR2 being catalytically active enzymes with distinct substrate preferences. Structural biology has been shown to be an important tool for understanding the basic mechanism and substrate specificity of ADARs. Information gleaned from structural studies can be used to optimize therapeutic site-directed RNA editing platforms, develop new genome editing technologies, and design ADAR inhibitor drugs for the treatment of cancer. Chapter 1 of this dissertation begins with an overview of the various modifications that occur in RNA, with an emphasis on adenosine-to-inosine RNA editing by ADARs. Next, I discuss the mechanism and substrate specificity of ADARs, the diverse biological implications of ADAR editing, and how aberrant editing is implicated in several human diseases. I go on to discuss how ADARs have been used to develop therapeutic site-directed RNA editing technologies. Lastly, I finish with a discussion of the structural information of ADARs to date and how these structures have provided important mechanistic and substrate specificity information. Chapters 2 and 3 describe highly collaborative studies to optimize guide oligonucleotide design for the site-directed RNA editing with endogenous ADARs. Chapter 2 examines modified nucleotides at the orphan base position to enhance on-target editing by endogenous ADARs. Benner’s base dZ was shown to enhance ADAR base-flipping, catalysis, and on-target editing by wild type ADAR. A high-resolution crystal structure of the deaminase domain of ADAR

The respiratory disease tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis (Mtb), is an ongoing worldwide epidemic that necessitates the identification of novel drug targets. Sulfur is an essential element for the growth, virulence, and survival of Mtb, and thus disruption of sulfur metabolism may be a potential means of combating TB. This dissertation discusses research conducted on CysDNC, the key sulfate activating complex (SAC) that lies at the start of the mycobacterial sulfur pathway. Sulfur metabolism begins when intracellular sulfate is reacted with adenosine 5′-triphosphate (ATP) to form the product adenosine 5’-phosphosulfate (APS). This reaction is catalyzed by the ATP sulfurylase CysD and is energetically coupled to hydrolysis of GTP by the GTPase domain of CysNC. APS is then further phosphorylated into 3’-phosphoadenosine-5’-phosphosulfate (PAPS) by the APS kinase domain of CysNC. The products APS and PAPS serve as precursors for downstream sulfur-containing biomolecules. The individual subunits CysD and CysNC associate together to form the trifunctional CysDNC complex. In this dissertation, CysDNC has been characterized by several approaches.The opening chapter of this work reviews the role of sulfur-containing compounds in TB infection, previous work done on bacterial SACs, and sulfate-activating enzymes from various species across the kingdom of life. Chapter 2 proceeds with initial observations on the structure and stability of CysDNC during recombinant purification of the complex from the expression host Escherichia coli as well as M. smegmatis, a species closely related to M. tuberculosis. Kinetic studies of CysDNC are discussed in Chapter 3. The activity of CysDNC in the reverse direction of the ATP sulfurylase reaction was characterized in the absence and presence of GTP and GDP. Two other kinetic assays, a commercially-available pyrophosphate detection assay and a “molybdolysis” assay that aimed to decouple the ATP sulfurylase

Due to population growth, shifts in land use, and climate change, there is an increasing demand for fresh water worldwide. Groundwater is an increasingly critical resource used to meet demand but is facing both decreasing supply and quality. Many aquifers have the capacity to store more groundwater, and managed aquifer recharge (MAR), in which surface water in infiltrated into shallow aquifers for later use, is a strategy that can help to replenish groundwater supplies and potentially improve water quality. In order to ensure that MAR systems deliver maximum benefits, it is critical to quantify the hydrologic and biogeochemical processes that occur during infiltration. In this dissertation, I present three original studies that use field, laboratory, and analytical techniques to investigate the linkages between the hydrologic and biogeochemical cycles during infiltration for MAR and the potential implications for groundwater management. In Chapter One, plot-scale infiltration tests showed that a readily-available soil carbon amendment (wood chips) could enhance nitrate removal during rapid infiltration. In Chapter 2, linked field and laboratory studies demonstrated that another soil carbon amendment, almond shells, can promote more efficient nitrate removal than wood chips, but also can facilitate the release of geogenic trace metals. In Chapter Three, multi-criteria decision analysis (MCDA) was paired with a GIS in order to assess where recharge occurs, could occur, or could be developed through MAR projects in Santa Clara County, California. Collectively, these studies help to further our understanding of both hydrologic and biogeochemical processes and inform sustainable water resource management.

In this study, we highlight the importance of permeability anisotropy on the hydrogeological regime of a ridge-flank hydrothermal system. Our study site, North Pond, is a marine sediment pond on ∼8 Ma seafloor in the North Atlantic, and represents a low-temperature, end-member ridge-flank hydrothermal system. Previous simulations of North Pond elucidated long-standing hypotheses concerning hydrothermal fluid and heat transport in the upper volcanic crust but failed to fully explain observed patterns of seafloor heat flux in this area. Here we use variography, a geostatistical method, to quantify relations between seafloor heat-flux measurements, and coupled numerical simulations of fluid and heat flow to simulate the hydrogeologic regime. Directional variography shows that heat-flux observations are correlated along-strike of the regional crustal fabric. Three-dimensional simulations that include permeability anisotropy are able to replicate seafloor heat-flux patterns across North Pond. The simulations that result in the best match to thermal data incorporate permeability anisotropy in the horizontal plane. We find that the feedback between permeability anisotropy and the asymmetric geometry of North Pond combine to promote advective removal of heat and mass within the crustal aquifer. These findings suggest that permeability anisotropy in the oceanic crust may influence ridge-flank hydrothermal circulation more broadly.

Leading deep-sea research expeditions requires a breadth of training and experience, and the opportunities for Early Career Researchers (ECRs) to obtain focused mentorship on expedition leadership are scarce. To address the need for leadership training in deep-sea expeditionary science, the Crustal Ocean Biosphere Research Accelerator (COBRA) launched a 14-week virtual Master Class with both synchronous and asynchronous components to empower students with the skills and tools to successfully design, propose, and execute deep-sea oceanographic field research. The Master Class offered customized and distributed training approaches and created an open-access syllabus with resources, including reading material, lectures, and on-line resources freely-available on the Master Class website (cobra.pubpub.org). All students were Early Career Researchers (ECRs, defined here as advanced graduate students, postdoctoral scientists, early career faculty, or individuals with substantial industry, government, or NGO experience) and designated throughout as COBRA Fellows. Fellows engaged in topics related to choosing the appropriate deep-sea research asset for their Capstone “dream cruise” project, learning about funding sources and how to tailor proposals to meet those source requirements, and working through an essential checklist of pre-expedition planning and operations. The Master Class covered leading an expedition at sea, at-sea operations, and ship-board etiquette, and the strengths and challenges of telepresence. It also included post-expedition training on data management strategies and report preparation and outputs. Throughout the Master Class, Fellows also discussed education and outreach, international ocean law and policy, and the importance and challenges of team science. Fellows further learned about how to develop concepts respectfully with regard to geographic and cultural considerations of their intended study sites. An assessment of initial outcomes from the fir

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and bears multiple spike mutations2. Here we show that Omicron spike has higher affinity for ACE2 compared to Delta as well as a marked change of antigenicity conferring significant evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralising antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralisation. Importantly, antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lower airway organoids, lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared to Delta. Replication differences mapped to entry efficiency using spike pseudotyped virus (PV) assays. The defect for Omicron PV to enter specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and knock down of TMPRSS2 impacted Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently utilises the cellular protease TMPRSS2 that promotes cell entry via plasma membrane fusion, with greater dependency on cell entry via the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to utilise TMPRSS2, syncytium formation by the Omicron spike was markedly impaired compared to the Delta spike. Omicron’s less efficient spike cleavage at S1/S2 is associated with shift in cellular tropism away from TMPRSS2 expressing cells, with implications for altered pathogenesis.

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in German, C., Blackman, D., Fisher, A., Girguis, P., Hand, K., Hoehler, T., Huber, J., Marshall, J., Pietro, K., Seewald, J., Shock, E., Sotin, C., Thurnherr, A., & Toner, B. Ocean system science to inform the exploration of ocean worlds. Oceanography, 35(1), (2022): 16-20, https://doi.org/10.5670/oceanog.2021.411., Ocean worlds provide fascinating opportunities for future ocean research. They allow us to test our understanding of processes we consider fundamental to Earth’s ocean and simultaneously provide motivation to explore our ocean further and develop new technologies to do so. In parallel, ocean worlds research offers opportunities for ocean scientists to provide meaningful contributions to novel investigations in the coming decades that will search for life beyond Earth. Key to the contributions that oceanographers can make to this field is that studies of all other ocean worlds remain extremely data limited. Here, we describe an approach based on ocean systems science in which theoretical modeling can be used, in concert with targeted laboratory experimentation and direct observations in Earth’s ocean, to predict what processes (including those essential to support life) might be occurring on other ocean worlds. In turn, such an approach would help identify new technologies that might be required for future space missions as well as appropriate analog studies that could be conducted on Earth to develop and validate such technologies. Our approach is both integrative and interdisciplinary and considers multiple domains, from processes active in the subseafloor to those associated with ocean-ice feedbacks., This work is supported by NASA Astrobiology Program award # 80NSSC19K1427 Exploring Ocean Worlds: Ocean System Science to Support the Search for Life.

Ridge-flank hydrothermal circulation is a critical crustal process that affects the Earth's global heat budget, geochemical signature of the Earth's oceans, and supports a vast and diverse subsurface biosphere. These hydrothermal systems are globally pervasive, found in ocean crust of varying ages, spreading rates, and depths. In addition to the broad spatial and structural occurrence, ridge-flank hydrothermal systems operate on a wide range of temperatures, some barely above the temperature of bottom sea water and others near boiling. This dissertation focuses on North Pond, an important end-member, ridge-flank, cool hydrothermal system located on slow-spreading crust in the North Atlantic Ocean. Here I present three original studies that elucidate hydrogeologic and thermal characteristics of North Pond. In chapter 1, I present the first coupled three-dimensional simulations of fluid and heat below North Pond revising many long-standing hypotheses in this system. In chapter 2, I extend three-dimensional simulations to incorporate anisotropic permeability representing important crustal processes and provide a more holistic approach to characterizing the hydrogeologic and thermal regimes of North Pond. Finally, in chapter 3, I develop a workflow for constructing geologically realistic simulation domains and provide insights and rationale about important considerations during this process. The combination of these studies sheds light on the hydrogeological and thermal regime of this non-discharge dominated, end-member system and provides insights into the drivers of hydrological and thermal processes that are important across the spectrum of ridge-flank hydrothermal systems.

Adenosine deaminases acting on RNA (ADAR) are a family of enzymes responsible for the conversion of adenosine to inosine in double stranded RNA. Inosine selectively base pairs with cytidine and is recognized like guanosine by cellular machinery. A-to-I editing has a myriad of downstream effects and dysregulated editing by ADARs is observed in multiple neurological and oncogenic diseases. In humans there are three ADAR enzymes, named ADAR1, ADAR2 and ADAR3, each with their own unique polypeptide sequences and RNA substrate preferences. There is great interest in uncovering the structural basis of RNA editing by ADAR with the potential to accelerate the development of targeted therapeutics to restore homeostatic levels of RNA editing in diseased patients. This thesis describes the effort to understand the structural basis behind ADAR and RNA interactions. Chapter 1 will provide an overview of what is known about RNA editing and describe the ways in which it is implicated in human disease. Chapter 2 describes the structural elucidation of an asymmetric protein-protein dimer of ADAR and how its formation aids in forming an RNA-bound complex. The biophysical characteristics of this protein-protein dimer will also be discussed, in addition to the identification of key conserved amino acid residues that are important for the formation of the protein-protein dimer. Chapter 3 describes the work towards the structural elucidation of the deaminase domain of ADAR1 bound to double stranded RNA. Current strategies to solve the crystal structure of ADAR1 bound to RNA will be discussed. A low resolution data set was collected. Information gained from the low-resolution model, how it was generated and strategies to optimize crystallization and diffraction to obtain a high-quality diffraction dataset will also be discussed. Chapter 4 will describe structural studies of ADAR2’s deaminase domain and an RNA duplex with a non-ideal nearest neighbor. Additionally, this chapter will discus

ADARs (adenosine deaminases acting on RNA) can be directed to sites in the transcriptome by complementary guide strands allowing for the correction of disease-causing mutations at the RNA level. However, ADARs show bias against editing adenosines with a guanosine 5' nearest neighbor (5'-GA sites), limiting the scope of this approach. Earlier studies suggested this effect arises from a clash in the RNA minor groove involving the 2-amino group of the guanosine adjacent to an editing site. Here we show that nucleosides capable of pairing with guanosine in a syn conformation enhance editing for 5'-GA sites. We describe the crystal structure of a fragment of human ADAR2 bound to RNA bearing a G:G pair adjacent to an editing site. The two guanosines form a Gsyn:Ganti pair solving the steric problem by flipping the 2-amino group of the guanosine adjacent to the editing site into the major groove. Also, duplexes with 2'-deoxyadenosine and 3-deaza-2'-deoxyadenosine displayed increased editing efficiency, suggesting the formation of a Gsyn:AH+anti pair. This was supported by X-ray crystallography of an ADAR complex with RNA bearing a G:3-deaza dA pair. This study shows how non-Watson-Crick pairing in duplex RNA can facilitate ADAR editing enabling the design of next generation guide strands for therapeutic RNA editing.

Introduction: The intraosseous (IO) route is one of the primary means of vascular access in critically ill and injured patients. The most common sites used are the proximal humerus, proximal tibia, and sternum. Sternal IO placement remains an often-overlooked option in emergency and prehospital medicine. Due to the conflicts in Afghanistan and Iraq the use of sternal IOs have increased.Methods: The authors conducted a limited review, searching PubMed and Google Scholar databases for “sternal IO,” “sternal intraosseous,” and “intraosseous” without specific date limitations. A total of 47 articles were included in this review.Results: Sternal IOs are currently FDA approved for ages 12 and older. Sternal IO access offers several anatomical, pharmacokinetic, hemodynamic, and logistical advantages over peripheral intravenous and other IO points of access. Sternal IO use carries many of the same risks and limitations as the humeral and tibial sites. Sternal IO gravity flow rates are sufficient for transfusing blood and resuscitation. In addition, studies demonstrated they are safe during active CPR.Conclusion: The sternal IO route remains underutilized in civilian settings. When considering IO vascular access in adults or older children, medical providers should consider the sternum as the recommended IO access, particularly if the user is a novice with IO devices, increased flow rates are required, the patient has extremity trauma, or administration of a lipid soluble drug is anticipated.

Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in double stranded RNA. Human ADARs can be directed to predetermined target sites in the transcriptome by complementary guide strands, allowing for the correction of disease-causing mutations at the RNA level. Here we use structural information available for ADAR2-RNA complexes to guide the design of nucleoside analogs for the position in the guide strand that contacts a conserved glutamic acid residue in ADARs (E488 in human ADAR2), which flips the adenosine into the ADAR active site for deamination. Mutating this residue to glutamine (E488Q) results in higher activity because of the hydrogen bond donating ability of Q488 to N3 of the orphan cytidine on the guide strand. We describe the evaluation of cytidine analogs for this position that stabilize an activated conformation of the enzyme-RNA complex and increase catalytic rate for deamination by the wild-type enzyme. A new crystal structure of ADAR2 bound to duplex RNA bearing a cytidine analog revealed a close contact between E488, stabilized by an additional hydrogen bond and altered charge distribution when compared to cytidine. In human cells and mouse primary liver fibroblasts, this single nucleotide modification increased directed editing yields when compared to an otherwise identical guide oligonucleotide. Our results show that modification of the guide RNA can mimic the effect of hyperactive mutants and advance the approach of recruiting endogenous ADARs for site-directed RNA editing.

All messenger RNAs in eukaryotes are modified co-transcriptionally and post-transcriptionally. They are all capped at the 5'-end and polyadenylated at the 3'-end. However, many mRNAs are also found to be chemically modified internally for regulation of mRNA processing, translation, stability, and to recode the message. This review will briefly summarize the structural basis for formation of the two most common modifications found at internal sites in mRNAs; methylation and deamination. The structures of the enzymes that catalyze these modifications show structural similarity to other family members within each modifying enzyme class. RNA methyltransferases, including METTL3/METTL14 responsible for N6-methyladensosine (m6A) formation, share a common structural core and utilize S-adenosyl methionine as a methyl donor. RNA deaminases, including adenosine deaminases acting on RNA (ADARs), also share a common structural core and similar signature sequence motif with conserved residues used for binding zinc and catalyzing the deamination reaction. In spite of recent reports of high resolution structures for members of these two RNA-modifying enzyme families, a great deal remains to be uncovered for a complete understanding of the structural basis for mRNA modification. Of particular interest is the definition of factors that control modification site specificity.

Nipo Strongheart's role in the DeMille production of Braveheart enables us to see the Native American performers of his generation as actors, in multiple senses of the word, not as mere objects of the cinematic gaze. Like other “contact zones” where Indigenous intellectuals and performers engage with non-Indigenous audiences and expectations, Native agency was exercised in the movie industry as well. This article demonstrates that even as they forged careers and identities in studio Hollywood, professionals such as Strongheart would negotiate the terms of Native representation and attempt to disrupt the dominant discourse.

Repetitive mild traumatic brain injury in American football players has garnered increasing public attention following reports of chronic traumatic encephalopathy, a progressive tauopathy. While the mechanisms underlying repetitive mild traumatic brain injury-induced neurodegeneration are unknown and antemortem diagnostic tests are not available, neuropathology studies suggest a pathogenic role for microvascular injury, specifically blood-brain barrier dysfunction. Thus, our main objective was to demonstrate the effectiveness of a modified dynamic contrast-enhanced MRI approach we have developed to detect impairments in brain microvascular function. To this end, we scanned 42 adult male amateur American football players and a control group comprising 27 athletes practicing a non-contact sport and 26 non-athletes. MRI scans were also performed in 51 patients with brain pathologies involving the blood-brain barrier, namely malignant brain tumours, ischaemic stroke and haemorrhagic traumatic contusion. Based on data from prolonged scans, we generated maps that visualized the permeability value for each brain voxel. Our permeability maps revealed an increase in slow blood-to-brain transport in a subset of amateur American football players, but not in sex- and age-matched controls. The increase in permeability was region specific (white matter, midbrain peduncles, red nucleus, temporal cortex) and correlated with changes in white matter, which were confirmed by diffusion tensor imaging. Additionally, increased permeability persisted for months, as seen in players who were scanned both on- and off-season. Examination of patients with brain pathologies revealed that slow tracer accumulation characterizes areas surrounding the core of injury, which frequently shows fast blood-to-brain transport. Next, we verified our method in two rodent models: rats and mice subjected to repeated mild closed-head impact injury, and rats with vascular injury inflicted by photothrombosis. In

Bronchoalveolar lavage (BAL) is a key clinical and research tool in lung transplantation (LTx). However, BAL collection and processing are not standardized across LTx centers. This International Society for Heart and Lung Transplantation-supported consensus document on BAL standardization aims to clarify definitions and propose common approaches to improve clinical and research practice standards. The following 9 areas are covered: (1) bronchoscopy procedure and BAL collection, (2) sample handling, (3) sample processing for microbiology, (4) cytology, (5) research, (6) microbiome, (7) sample inventory/tracking, (8) donor bronchoscopy, and (9) pediatric considerations. This consensus document aims to harmonize clinical and research practices for BAL collection and processing in LTx. The overarching goal is to enhance standardization and multicenter collaboration within the international LTx community and enable improvement and development of new BAL-based diagnostics.

The effective management of water resources faces unprecedented pressures from growing demand and shifting climate among other stressors. Widespread issues of both water quantity and water quality affect many regions. One common problem facing many areas is the excess supply of nitrate (NO3) to receiving bodies such as aquifers, rivers, streams, and estuaries. Excess nitrate is linked to human activity and can have detrimental effects of human and ecosystem health. Addressing these issues and ensuring that humans and ecosystems have sufficient supplies of clean water requires a more complete understanding of the complex linkages between water quantity and quality across space and time. In this dissertation, I present four original studies across a range of scales investigating the flow of water across and through landscapes that have been altered by human activity. In each study I ask the fundamental question how does the magnitude, timing, and/or rate of water flow affect key water quality parameters? Chapters One, Two, and Three focus on investigating water quality improvements during managed aquifer recharge (MAR), a technique for improving groundwater supply and quality through targeted infiltration. In Chapter One, measurements of water quality parameters during controlled percolation experiments revealed that soil amendments could enhance nitrate removal during infiltration. In Chapter Two, column studies conducted on soil cores collected from MAR sites demonstrated that the rate of infiltration and the presence of a soil amendment were key factors controlling nitrate removal and affecting microbial community composition during MAR. In Chapter 3, we synthesized data from various scales to develop a novel, spatial model of nitrate removal during MAR based on soil and fluid properties. Finally, in Chapter Four, similar concepts are applied to the analysis of nitrate mobilization in streams and rivers of agricultural watersheds to analyze the patterns and timing

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2.

Bacterial nonhydrolyzing UDP-N-acetylglucosamine 2-epimerases catalyze the reversible interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). UDP-ManNAc is an important intermediate in the biosynthesis of certain cell-surface polysaccharides, including those in some pathogenic bacteria, such as Neisseria meningitidis and Streptococcus pneumoniae. Many of these epimerases are allosterically regulated by UDP-GlcNAc, which binds adjacent to the active site and is required to initiate UDP-ManNAc epimerization. Here, two crystal structures of UDP-N-acetylglucosamine 2-epimerase from Neisseria meningitidis serogroup A (NmSacA) are presented. One crystal structure is of the substrate-free enzyme, while the other structure contains UDP-GlcNAc substrate bound to the active site. Both structures form dimers as seen in similar epimerases, and substrate binding to the active site induces a large conformational change in which two Rossmann-like domains clamp down on the substrate. Unlike other epimerases, NmSacA does not require UDP-GlcNAc to instigate the epimerization of UDP-ManNAc, although UDP-GlcNAc was found to enhance the rate of epimerization. In spite of the conservation of residues involved in binding the allosteric UDP-GlcNAc observed in similar UDP-GlcNAc 2-epimerases, the structures presented here do not contain UDP-GlcNAc bound in the allosteric site. These structural results provide additional insight into the mechanism and regulation of this critical enzyme and improve the structural understanding of the ability of NmSacA to epimerize modified substrates.

Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.

2020, 2020 ISAZ. The welfare of aquatic animals is receiving increasing attention around the world, and this should be reflected in the veterinary curricular. Veterinary students in Australia and New Zealand ranked the importance of five pre-selected topics on aquatic animal welfare and ethical issues for newly qualified veterinarians in an online survey. They ranked aquatic animals' health and welfare issues, husbandry techniques of farmed fish, and the use of antibiotics as being of high importance for their first day in practice, with pain and distress associated with angling and trawling practices (fishing) and euthanasia ranking moderate to high. Females assigned more importance to fishing, health and welfare issues, and husbandry techniques of farmed fish than did males. Students in the early stages of study assigned more importance to fishing than did senior students. To the authors' knowledge, this is the first study to investigate what importance veterinary students place on their competence regarding aquatic animal welfare and ethics. Gaining an understanding of how veterinary students prioritize these issues, and if this differs based on gender and stage of study, will help veterinary educators to better prepare graduates to address concerns surrounding the welfare of aquatic animals. This could be achieved by careful consideration of course content and where in the curriculum this material is taught, as well as reiterating key messages in the senior years and addressing changing student attitudes as they progress through the course.

Molecules capable of directing changes to nucleic acid sequences are powerful tools for molecular biology and promising candidates for the therapeutic correction of disease-causing mutations. However, unwanted reactions at off-target sites complicate their use. Here we report selective combinations of mutant editing enzyme and directing oligonucleotide. Mutations in human ADAR2 (adenosine deaminase acting on RNA 2) that introduce aromatic amino acids at position 488 reduce background RNA editing. This residue is juxtaposed to the nucleobase that pairs with the editing site adenine, suggesting a steric clash for the bulky mutants. Replacing this nucleobase with a hydrogen atom removes the clash and restores editing activity. A crystal structure of the E488Y mutant bound to abasic site-containing RNA shows the accommodation of the tyrosine side chain. Finally, we demonstrate directed RNA editing in vitro and in human cells using mutant ADAR2 proteins and modified guide RNAs with reduced off-target activity.

The Internet of Things (IoT) age is upon us. As we look to build larger networks with more devices connected to the Internet, the need for lightweight protocols that minimize the use of both energy and computation gain popularity. One such protocol is Message Queue Telemetry Transport (MQTT). Since its introduction in 1999, it has slowly increased in use cases and gained a huge spike in popularity since it was used in the popular messaging application Facebook Messenger. In our previous works, we focused on adding geolocation to MQTT, to help modernize the protocol into the IoT age. In this paper, we build off our previous work on MQTTg and build an IoT Android Application that can pull geolocation information from the Operating System. We then use the geolocation data to create geofences to help further tailor the use cases of MQTTg., Comment: 5 pages, 6 figures. arXiv admin note: substantial text overlap with arXiv:1811.09706

Groundwater is a critical resource facing declining supply and quality around the world. Distributed stormwater collection coupled with managed aquifer recharge (DSC-MAR) is a developing strategy to enhance groundwater recharge by collecting excess hillslope runoff in infiltration basins. DSC-MAR projects provide opportunities to learn about the inter-related hydrologic processes of runoff generation, infiltration, and recharge in the context of sustainable groundwater management. The three chapters presented here use interdisciplinary approaches to study these processes at multiple scales, including (1) regional computer models evaluating the impact of shifting climate and land use on runoff generation and recharge, (2) multi-year field observations quantifying the dynamics of runoff generation and infiltration, and (3) field-based experiments linking hydrologic, geochemical, and microbial processes during rapid infiltration. In Chapter 1, models suggest that urban and agricultural development reduce the threshold for runoff generation relative to pre-development land use, resulting in up to 2.3 times as much runoff and less recharge basin-wide. When incorporated into an MAR suitability map combining soil, bedrock, and aquifer properties, model results demonstrate that DSC-MAR projects could be effective in many locations during a range of climate scenarios. Field results from Chapter 2 show that a pilot DSC-MAR project was successful, collecting and infiltrating 5.3 × 105 m3 (426 ac-ft) over six years, including an extended regional drought. Runoff generation was sensitive to sub-daily storm frequency, duration, and intensity; observed infiltration rates varied widely in space and time; and 8.2 × 105 kg of fine-grained sediment accumulated in the infiltration basin over three years, likely reducing soil infiltration capacity. In Chapter 3, soils below a horizontal permeable reactive barrier (PRB) made of woodchips had greater nitrate removal (1.5 g/m2/day NO3-N) t

This issue brief considers whether groundwater recharge currently qualifies, or should qualify, as a beneficial use of surface water under a California water right. Currently, the lack of an explicit policy regarding recharge for non-extractive purposes — that is, for purposes such as combatting subsidence, raising regional groundwater levels, or supporting baseflow or ground-water dependent wetlands — creates uncertainty and confusion. To bring much needed clarity, the State Water Resources Control Board (the Board) should provide guidance explaining that recharge for non-extractive purposes can be a beneficial use of water. That guidance should explain the conditions under which recharge for nonextractive purposes is beneficial and the evidence water managers should provide to support a beneficial use determination.

Groundwater sustainability depends on balancing aquifer inflows and outflows. Extraction (pumping of groundwater, typically for human use) and recharge (inflow of water to an aquifer from the land surface and streams) are central components of this water balance. Often, increasing demands for groundwater are exacerbated by stresses on limited surface water supplies. Changes in land use and shifting climate can result in less infiltration of precipitation into the ground, reducing recharge. Increasing water scarcity has led to increased pumping, and in turn, unsustainable management of groundwater in many basins, resulting in depleted supplies, degraded water quality, and other impacts. Conservation strategies have reduced demand in some basins, and there are also opportunities for increasing recharge; both strategies can help to tip the water balance towards sustainability. Natural recharge occurs across the landscape, in forests and fields, and below rivers and streams; it is a fundamental hydrologic process that is difficult to measure or control because it varies so greatly in location and timing. Managed aquifer recharge (MAR) is a set of techniques used to improve groundwater conditions by routing more surface water into aquifers. MAR can be applied at many scales, from street corner swales to regional systems. MAR based on the distributed collection of stormwater (“distributed MAR”) can be accomplished at an intermediate scale, generating hundreds to thousands of acre-feet/year of infiltration benefit. The promise of distributed MAR stems from its modest cost, and comparative simplicity of design and operation. Distributed MAR projects can be developed on private or public land across a groundwater basin, potentially generating more total benefit than smaller scale installations, and with less cost and complexity than regional MAR systems. A key challenge for developing distributed MAR projects lies in creating incentives that will motivate landowners, tenants

With California’s drought risk, flood risk, and demand for water all increasing, effective monitoring is more important than ever to water decision making. Stream gages monitor the most basic vital sign of California’s waterways—stream flow.1 Stream flow data support day-to-day decisions about how to manage water and operate water infrastructure. In turn, those decisions have important implications for flood control and the water supplies upon which residential, industrial, agricultural, and environmental water users depend. Stream flow information also provides technical insights into basin hydrology, and those insights aid long-term water planning. As pressures on the state’s water systems intensify, the need for accurate and timely stream flow information will continue to grow. Opportunities for better water management created by groundbreaking legislation such as the Sustainable Groundwater Management Act (SGMA), the Open and Transparent Water Data Act, and Proposition 1 will be severely limited without an effective network of continuous stream gaging.

Effectors are microbial-derived secreted proteins with an essential function in modulating host immunity during infections. CfAvr4, an effector protein from the tomato pathogen Cladosporium fulvum and the founding member of a fungal effector family, promotes parasitism through binding fungal chitin and protecting it from chitinases. Binding of Avr4 to chitin is mediated by a carbohydrate-binding module of family 14 (CBM14), an abundant CBM across all domains of life. To date, the structural basis of chitin-binding by Avr4 effector proteins and of recognition by the cognate Cf-4 plant immune receptor are still poorly understood. Using X-ray crystallography, we solved the crystal structure of CfAvr4 in complex with chitohexaose [(GlcNAc)6] at 1.95Å resolution. This is the first co-crystal structure of a CBM14 protein together with its ligand that further reveals the molecular mechanism of (GlcNAc)6 binding by Avr4 effector proteins and CBM14 family members in general. The structure showed that two molecules of CfAvr4 interact through the ligand and form a three-dimensional molecular sandwich that encapsulates two (GlcNAc)6 molecules within the dimeric assembly. Contrary to previous assumptions made with other CBM14 members, the chitohexaose-binding domain (ChBD) extends to the entire length of CfAvr4 with the reducing end of (GlcNAc)6 positioned near the N-terminus and the non-reducing end at the C-terminus. Site-directed mutagenesis of residues interacting with (GlcNAc)6 enabled the elucidation of the precise topography and amino acid composition of Avr4's ChBD and further showed that these residues do not individually mediate the recognition of CfAvr4 by the Cf-4 immune receptor. Instead, the studies highlighted the dependency of Cf-4-mediated recognition on CfAvr4's stability and resistance against proteolysis in the leaf apoplast, and provided the evidence for structurally separating intrinsic function from immune receptor recognition in this effector family.

Plants employ cell-surface pattern recognition receptors (PRRs) to detect pathogens. Although phytohormones produced during PRR signaling play an essential role in innate immunity, a direct link between PRR activation and hormone regulation is unknown. EFR is a PRR that recognizes bacterial EF-Tu and activates immune signaling. Here we report that EFR regulates the phytohormone jasmonic acid (JA) through direct phosphorylation of a receptor-like cytoplasmic kinase, BIK1. The BIK1 structure revealed that the EFR-phosphorylated sites reside on a uniquely extended loop away from the BIK1 kinase core domain. Phosphomimetic mutations of these sites resulted in increased phytohormones and enhanced resistance to bacterial infections. In addition to its documented plasma membrane localization, BIK1 also localizes to the nucleus and interacts directly with WRKY transcription factors involved in the JA and salicylic acid (SA) regulation. These findings demonstrate the mechanistic basis of signal transduction from PRR to phytohormones, mediated through a PRR-BIK1-WRKY axis.

Green communications is the practice of selecting energy efficient communications, networking technologies and products. This process is followed by minimizing resource use whenever possible in all branches of communications. In this day and age, green communication is vital to the footprint we leave on this planet as we move into a completely digital age. One such communication tool is Message Queue Transport Telemetry or MQTT which is an open source publisher/subscriber standard for M2M (Machine to Machine) communication. It is well known for its low energy and bandwidth footprint and thus makes it highly suitable for Green Internet of Things (IoT) messaging situations where power usage is at a premium or in mobile devices such as phones, embedded computers or microcontrollers. It is a perfect tool for the green communication age upon us and more specifically Green IoT. One problem however with the original MQTT protocol is that it is lacking the ability to broadcast geolocation. In today's age of IoT however, it has become more pertinent to have geolocation as part of the protocol. In this paper, we add geolocation to the MQTT protocol and offer a revised version, which we call MQTTg. We describe the protocol here and show where we are able to embed geolocation successfully. We also offer a glimpse into an Android OS application we are developing for Open Source use., Comment: 14 pages, 5 figures