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We describe the efficient creation of single-component optogenetic tools for membrane recruitment-based signaling perturbation using BcLOV4 technology. The workflow requires two plasmids to create six different domain arrangements of the dynamic membrane binder BcLOV4, a fluorescent reporter, and the fused signaling protein of interest. Screening of this limited set of genetic constructs for expression characteristics and dynamic translocation in response to one pulse of light is sufficient to identify viable signaling control tools. The reliability of this streamlined approach is demonstrated by the creation of an optogenetic Cdc42 GTPase and Rac1-activating Tiam1 GEF protein, which together with our other recently reported technologies, completes a toolbox for spatiotemporally precise induction of Rho-family GTPase signaling at the GEF or GTPase level, for driving filopodial protrusions, lamellipodial protrusions, and cell contractility, respectively mediated by Cdc42, Rac1, and RhoA.

Proteins often interconvert between different conformations in ways critical to their function. Although manipulating such equilibria for biophysical study is often challenging, the application of pressure is a potential route to achieve such control by favoring the population of lower volume states. Here, we use this feature to study the interconversion of ARNT PAS-B Y456T, which undergoes a dramatic +3 slip in the β-strand register as it switches between two stably folded conformations. Using high-pressure biomolecular NMR approaches, we obtained the first, to our knowledge, quantitative data testing two key hypotheses of this process: the slipped conformation is both smaller and less compressible than the wild-type equivalent, and the interconversion proceeds through a chiefly unfolded intermediate state. Data collected in steady-state pressure and time-resolved pressure-jump modes, including observed pressure-dependent changes in the populations of the two conformers and increased rate of interconversion between conformers, support both hypotheses. Our work exemplifies how these approaches, which can be generally applied to protein conformational switches, can provide unique information that is not easily accessible through other techniques.

Inducible gene expression systems are valuable tools for studying biological processes. We previously developed an optogenetic gene expression system called TAEL that is optimized for use in zebrafish. When illuminated with blue light, TAEL transcription factors dimerize and activate gene expression downstream of the TAEL-responsive C120 promoter. By using light as the inducing agent, the TAEL/C120 system overcomes limitations of traditional inducible expression systems by enabling fine spatial and temporal regulation of gene expression. Here, we describe ongoing efforts to improve the TAEL/C120 system. We made modifications to both the TAEL transcriptional activator and the C120 regulatory element, collectively referred to as TAEL 2.0. We demonstrate that TAEL 2.0 consistently induces higher levels of reporter gene expression and at a faster rate, but with comparable background and toxicity as the original TAEL system. With these improvements, we were able to create functional stable transgenic lines to express the TAEL 2.0 transcription factor either ubiquitously or with a tissue-specific promoter. We demonstrate that the ubiquitous line in particular can be used to induce expression at late embryonic and larval stages, addressing a major deficiency of the original TAEL system. This improved optogenetic expression system will be a broadly useful resource for the zebrafish community.

Recent research on fold-switching metamorphic proteins has revealed some notable exceptions to Anfinsen's hypothesis of protein folding. We have previously described how a single point mutation can enable a well-folded protein domain, one of the two PAS (Per-ARNT-Sim) domains of the human ARNT (aryl hydrocarbon receptor nuclear translocator) protein, to interconvert between two conformers related by a slip of an internal β strand. Using this protein as a test case, we advance the concept of a “fragile fold”, a protein fold that can reversibly rearrange into another fold that differs by a substantial number of hydrogen bonds, entailing reorganization of single secondary structure elements to more drastic changes seen in metamorphic proteins. Here we use a battery of biophysical tests to examine several factors affecting the equilibrium between the two conformations of the switching ARNT PAS-B Y456T protein. Of note is that we find that factors which impact the HI loop preceding the shifted Iβ strand affect both the equilibrium levels of the two conformers and the denatured state which links them in the interconversion process. Finally, we describe small molecules that selectively bind to and stabilize the wild-type conformation of ARNT PAS-B. These studies form a toolkit for studying fragile protein folds and could enable ways to modulate the biological functions of such fragile folds, both in natural and engineered proteins.

Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light, oxygen, voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output partner. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 μs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in loss of the interaction between the side chain of N414 and the backbone C=O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

The notion of sustainable infrastructure for the delivery of social services is to fulfill basic human needs, in war-torn societies, human safety is a critical basic need. The relationship between sustainable infrastructure development and human safety remains underresearched in Afghan neighborhoods. Therefore, this study examined the effectiveness of the police facilities constructed for stability enhancement in Afghan communities. To do so, this study used Afghans&rsquo, polling datasets on the police presence and the public safety perceptions, including newly collected survey data related to the influence of the police facilities on human safety and other factors contributing to the neighborhoods&rsquo, well-being. The datasets are organized with a multilevel structure in which different individuals are sampled within neighborhoods and analyzed using a multilevel model approach to capture the randomness of the responses. The results showed that police facilities are more important to perceptions of safety in less safe areas and that Afghans in villages perceived themselves as safer than in urban areas, relative to their own immediate region. Those perceiving themselves as being safer were older, more highly educated, and widowed respondents. Overall, Afghans perceived the police facilities as institutional symbol for promoting improvements and opportunities for fulfilling basic human safety needs.

Metallic residues are distributed heterogeneously onto small-arms range soils from projectile fragmentation upon impact with a target or berm backstop. Incremental Sampling Methodology (ISM) can address the spatially heterogeneous contamination of surface soils on small-arms ranges, but representative kilogram-sized ISM subsamples are affected by the range of metallic residue particle sizes in the sample. This study compares the precision and concentrations of metals in a small-arms range soil sample processed by a puck mill, ring and puck mill, ball mill, and mortar and pestle prior to analysis. The ball mill, puck mill, and puck and ring mill produced acceptable relative standard deviations of less than 15% for the anthropogenic metals of interest (Lead (Pb), Antimony (Sb), Copper (Cu), and Zinc (Zn)), with the ball mill exhibiting the greatest precision for Pb, Cu, and Zn. Precision by mortar and pestle, without milling, was considerably higher (40% to >100%) for anthropogenic metals. Media anthropogenic metal concentrations varied by more than 40% between milling methods, with the greatest concentrations produced by the puck mill, followed by the puck and ring mill and then the ball mill. Metal concentrations were also dependent on milling time, with concentrations stabilizing for the puck mill by 300 s but still increasing for the ball mill over 20 h. Differences in metal concentrations were not directly related to the surface area of the milled sample. Overall, the tested milling methods were successful in producing reproducible data for soils containing metallic residues. However, the effects of milling type and time on concentrations require consideration in environmental investigations.

The general stress response (GSR) allows many bacterial species to react to myriad different stressors. In Alphaproteobacteria, this signaling pathway proceeds through the partner-switching PhyR-EcfG sigma-factor mechanism and is involved in multiple life processes, including virulence in Brucella abortus. To date, details of the alphaproteobacterial GSR signaling pathway have been determined using genetic and biochemical work on a diverse set of species distributed throughout the clade. Fiebig and co-workers establish Erythrobacter litoralis DSM 8509 as a genetically tractable lab strain and use it to both directly and indirectly delineate photoresponsive GSR pathways mediated by multiple HWE/HisKA_2 histidine kinases. The existence of a new phototrophic lab strain allows researchers to compare the GSR across different Alphaproteobacteria, as well as study the interplay between the GSR and phototrophy. Additionally, the discovery of new HWE/HisKA_2 kinases regulating the GSR poses new questions about how different stimuli feed into this widespread stress pathway.

Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light–sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme’s residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.

Recent research on fold-switching metamorphic proteins has revealed some notable exceptions to Anfinsen's hypothesis of protein folding. We have previously described how a single point mutation can enable a well-folded protein domain, one of the two PAS (Per-ARNT-Sim) domains of the human ARNT (aryl hydrocarbon receptor nuclear translocator) protein, to interconvert between two conformers related by a slip of an internal beta-strand. Using this protein as a test case, we advance the concept of a fragile fold, a protein fold that can reversibly rearrange into another fold that differs by a substantial number of hydrogen bonds, entailing reorganization of single secondary structure elements to more drastic changes seen in metamorphic proteins. Here we use a battery of biophysical tests to examine several factors affecting the equilibrium between the two conformations of the switching ARNT PAS-B Y456T protein. Of note, we find that factors which impact the HI loop preceding the shifted I(beta)-strand affect both the equilibrium levels of the two conformers and the denatured state which links them in the interconversion process. Finally, we describe small molecules that selectively bind to and stabilize the wildtype conformation of ARNT PAS-B. These studies form a toolkit for studying fragile protein folds and could enable ways to modulate the biological functions of such fragile folds, both in natural and engineered proteins.

In this issue of Structure,Shang and Kojetin (2021) present insights into the binding mechanism of artificial agonists to the PPARγ nuclear receptor. These data support a two-step model with induced fit and conformational selection aspects. This mechanism may exist in related receptors, providing new opportunities for drug development.

Small molecule binding within internal cavities provides a way to control protein function and structure, as exhibited in numerous natural and artificial settings. Unfortunately, most ways to identify suitable cavities require high-resolution structures a priori and may miss potential cryptic sites. Here we address this limitation via high-pressure solution NMR spectroscopy, taking advantage of the distinctive nonlinear pressure-induced chemical shift changes observed in proteins containing internal cavities and voids. We developed a method to rapidly characterize such nonlinearity among backbone 1H and 15N amide signals without needing to have sequence-specific chemical shift assignments, taking advantage of routinely available 15N-labeled samples, instrumentation, and 2D 1H/15N HSQC experiments. From such data, we find a strong correlation in the site-to-site variability in such nonlinearity with the total void volume within proteins, providing insights useful for prioritizing domains for ligand binding and indicating mode-of-action among such protein/ligand systems. We suggest that this approach provides a rapid and useful way to rapidly assess otherwise hidden dynamic architectures of protein that reflect fundamental properties associated with ligand binding and control.Significance StatementMany proteins can be regulated by internally binding small molecule ligands, but it is often not clear a priori which proteins are controllable in such a way. Here we describe a rapid method to address this challenge, using solution NMR spectroscopy to monitor the response of proteins to the application of high pressure. While the locations of NMR signals from most proteins respond to high pressure with linear chemical shift changes, proteins containing internal cavities that can bind small molecule ligands respond with easily identified non-linear changes. We demonstrate this approach on several proteins and protein/ligand complexes, suggesting that it has general utility.

Proteins often interconvert between different conformations in ways critical to their function. However, manipulating the equilibrium positions and kinetics of such conformational transitions has been traditionally challenging. Pressure is an effective thermodynamic variable for favoring the population of high energy protein conformational states with smaller volumes, as elegantly demonstrated in its use for biophysical studies of unfolding transitions. Here, we investigate the pressure-dependent effects of the interconversion of two stably-folded conformations of the Y456T variant of the aryl hydrocarbon receptor nuclear translocator (ARNT) Period/ARNT/Single-minded PAS-B domain. We previously discovered that ARNT PAS-B Y456T spontaneously interconverts between two structures primarily differing by a three-residue β-strand slip, inverting its topology in the process. This change collapses the internal cavities of the WT-like (WT) conformation of this protein as it adopts the slipped conformation (SLIP). Using pressure-NMR to conduct thermodynamic and kinetic analyses of the interconversion of ARNT PAS-B, we provide data that support two key predictions of this process: the SLIP conformation is both smaller and less compressible than the WT conformation, and the interconversion proceeds through a chiefly-unfolded intermediate state. We also find that the pressure-dependent NMR chemical shift changes and the residue-specific compressibility both predict which amino acid residues are near protein cavities. We demonstrate that pressure-NMR is a powerful approach for characterizing protein conformational switching and can provide unique information that is not easily accessible through other techniques.

Integral to the protein structure/function paradigm, oligomeric state is typically conserved along with function across evolution. However, notable exceptions such as the hemoglobins show how evolution can alter oligomerization to enable new regulatory mechanisms. Here we examine this linkage in histidine kinases (HKs), a large class of widely distributed prokaryotic environmental sensors. While the majority of HKs are transmembrane homodimers, members of the HWE/HisKA2 family can deviate from this architecture as exemplified by our finding of a monomeric soluble HWE/HisKA2 HK (EL346, a photosensing Light-Oxygen-Voltage (LOV)-HK). To examine the possibility of a wider range of possible relationships between oligomerization and regulation within this family, we biophysically and biochemically characterized multiple EL346 homologs and found a diverse range of HK oligomeric states and functions. Three LOV-HK homologs are dimers with differing structural and functional responses to light, while two are Per-ARNT-Sim (PAS)-HKs that interconvert between differentially active monomers and dimers, suggesting dimerization as a regulatory control. Finally, we examined putative interfaces in a dimeric LOV-HK, finding that multiple domains contributed to dimerization and function. Our findings suggest the potential for novel regulatory modes and oligomeric states beyond those traditionally defined for this important family of environmental sensors.

The New England region in the Northeast U.S. receives high annual precipitation as rain and snow, which results in floods that endanger people and infrastructure. Owing to the complexity of hydrolo...

Decisions about dams, like other environmental conflicts, involve complex trade-offs between different water uses with varying human and ecological impacts, have significant impacts on public resources, and involve many stakeholders with diverse and often conflicting interests. Given the many upcoming dam decisions in New England and across the United States, an improved understanding of public preferences about dam decisions is needed to steward resources in the public interest. This research asks (1) What does the public want to see happen with dams? and (2) How do public preferences regarding dam removal vary with demography and politics? We address these questions using data from three random sample statewide telephone polls conducted in New Hampshire over 2018 that asked people for their preferences concerning dam removal versus maintaining dams for specific benefits—property values, hydropower generation, industrial history, or recreation. Respondent age, education, gender, and political party were tested among the possible predictors. We find that majorities (52% or 54%) of respondents favor removing dams rather than keeping them for industrial history or property values, and a plurality (43%) favor removal over keeping them for recreation. A plurality (46%) prefer keeping dams, however, if they are used to generate hydropower. Respondent background characteristics and political identity affect these preferences in ways resembling those for many other environment-related issues: women, young or middle-aged individuals, and political liberals or moderates (Democrats or independents) more often support dam removal. Education, on the other hand, has no significant effects. The results quantify levels of general public support for dam removal in New England, illustrating the use of public opinion polling to complement input from public meetings and guide decisions. More broadly, they contribute a new topic to existing scholarship on the social bases of environmental concern.

Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.

Hydropower is traditionally considered to be one type of “clean” energy, and has been heavily developed in many regions of the world. Nevertheless, this assumption is increasingly being challenged by recent findings that a large amount of methane and other greenhouse gases (GHGs) are emitted during reservoir creation, turbine operation, and dam decommissioning. Via a critical review of existing hydropower life cycle assessments and reservoir emission studies, we compared the GHG emissions of various types of dams based on their structural type, size, primary function, and geographical location during their construction, operation, and decommissioning phases. Means to improve dam performance and reduce related GHG emissions were identified. It was found that dams with reservoirs usually have much higher GHG emission rates than diversion dams. GHG emissions are mainly generated at the construction and maintenance stages for small-scale run-of-river dams, whereas decomposition of flooded biomass and organic matter in the sediment has the highest GHG emission contribution to large-scale reservoir-based dams. Generally, reservoir-based dams located in boreal and temperate regions have much lower reservoir emissions (3–70 g CO2 eq./kW h) compared with dams located in tropical regions (8–6647 g CO2 eq./kW h). Our analysis shows that although most hydroelectric dams have comparable GHG emissions to other types of renewable energy (e.g., solar, wind energy), electricity produced from tropical reservoir-based dams could potentially have a higher emission rate than fossil-based electricity.

As drinking water supply systems plan for sustainable management practices, impacts from future water quality and climate changes are a major concern. This study aims to understand the intraannual changes of energy consumption for water treatment, investigate the relative importance of water quality and climate indicators on energy consumption for water treatment, and predict the effects of climate change on the embodied energy of treated, potable water at two municipal drinking water systems located in the northeast and southeast US. To achieve this goal, a life cycle assessment was first performed to quantify the monthly energy consumption in the two drinking water systems. Regression and relative importance analyses were then performed between climate indicators, raw water quality indicators, and chemical and energy usages in the treatment processes to determine their correlations. These relationships were then used to project changes in embodied energy associated with the plants' processes, and the results were compared between the two regions. The projections of the southeastern US water plant were for an increase in energy demand resulted from an increase of treatment chemical usages. The northeastern US plant was projected to decrease its energy demand due to a reduced demand for heating the plant's infrastructure. The findings indicate that geographic location and treatment process may determine the way climate change affects drinking water systems.

This study compares conventional grab sampling to incremental sampling methodology (ISM) to characterize metal contamination at a military small-arms-range. Grab sample results had large variances, positively skewed non-normal distributions, extreme outliers, and poor agreement between duplicate samples even when samples were co-located within tens of centimeters of each other. The extreme outliers strongly influenced the grab sample means for the primary contaminants lead (Pb) and antinomy (Sb). In contrast, median and mean metal concentrations were similar for the ISM samples. ISM significantly reduced measurement uncertainty of estimates of the mean, increasing data quality (e.g., for environmental risk assessments) with fewer samples (e.g., decreasing total project costs). Based on Monte Carlo resampling simulations, grab sampling resulted in highly variable means and upper confidence limits of the mean relative to ISM.

Membrane proteins, and especially G-protein coupled receptors (GPCRs), are increasingly important targets of structural biology studies due to their involvement in many biomedically-critical pathways in humans. These proteins are often highly dynamic and thus benefit from studies by NMR spectroscopy in parallel with complementary crystallographic and cryo-EM analyses. However, such studies are often complicated by a range of practical concerns, including challenges in preparing suitably isotopically-labeled membrane protein samples, large sizes of protein/detergent or protein/lipid complexes, and limitations on sample concentrations and stabilities. Here we describe our approach to addressing these challenges via the use of simple eukaryotic expression systems and modified NMR experiments, using the human adenosine A(2A) receptor as an example. Protocols are provided for the preparation of U-(2)H ((13)C,(1)H-Ile δ1) labeled membrane proteins from overexpression in the methylotrophic yeast Pichia pastoris, as well as techniques for studying the fast psec-nsec sidechain dynamics of the methyl groups of such samples. We believe that, with the proper optimization, these protocols should be generalizable to other GPCRs and human membrane proteins.

We evaluate the relative desirability of alternative futures for the upper Merrimack River watershed in New Hampshire, USA based on the value of ecosystem services at the end of the 21st century as gauged by its present-day inhabitants. This evaluation is accomplished by integrating land-use and socioeconomic scenarios, downscaled climate projections, biogeophysical simulation models, and the results of a citizen-stakeholder deliberative multicriteria evaluation. We find that although there are some trade-offs between alternative plausible futures, for the most part, it can be expected that future inhabitants of the watershed will be most satisfied if land-use planning in the intervening years prioritizes water supply and flood protection as well as maintenance of existing farmland and forest cover. With respect to climate change, it is expected that future watershed inhabitants will be more negatively affected by the projected loss of snow cover than the anticipated increase in hot summer days. More important than the specific results for the upper Merrimack River watershed, this integrative assessment demonstrates the complex yet ultimately informative potential to link stakeholder engagement with scenario generation, ecosystem models, and multiattribute evaluation for informing regional-scale planning and decision making.

The phototropins (phots) are light-activated kinases that are critical for plant physiology and the many diverse optogenetic tools that they have inspired. Phototropins combine two blue-light-sensing Light–Oxygen–Voltage (LOV) domains (LOV1 and LOV2) and a C-terminal serine/threonine kinase domain, using the LOV domains to control the catalytic activity of the kinase. While much is known about the structure and photochemistry of the light-perceiving LOV domains, particularly in how activation of the LOV2 domain triggers the unfolding of alpha helices that communicate the light signal to the kinase domain, many questions about phot structure and mechanism remain. Recent studies have made progress addressing these questions by utilizing small-angle X-ray scattering (SAXS) and other biophysical approaches to study multidomain phots from Chlamydomonas and Arabidopsis, leading to models where the domains have an extended linear arrangement, with the regulatory LOV2 domain contacting the kinase domain N-lobe. We discuss this and other advances that have improved structural and mechanistic understanding of phot regulation in this review, along with the challenges that will have to be overcome to obtain high-resolution structural information on these exciting photoreceptors. Such information will be essential to advancing fundamental understanding of plant physiology while enabling engineering efforts at both the whole plant and molecular levels.

Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1β) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1β, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.

Summary Information transmission in biological signaling networks is commonly considered to be a unidirectional flow of information between protein partners. According to this view, many bacterial response regulator proteins utilize input receiver (REC) domains to "switch" functional outputs, using REC phosphorylation to shift pre-existing equilibria between inactive and active conformations. However, recent data indicate that output domains themselves also shift such equilibria, implying a "mutual inhibition" model. Here we use solution nuclear magnetic resonance to provide a mechanistic basis for such control in a PhyR-type response regulator. Our structure of the isolated, non-phosphorylated REC domain surprisingly reveals a fully active conformation, letting us identify structural and dynamic changes imparted by the output domain to inactivate the full-length protein. Additional data reveal transient structural changes within the full-length protein, facilitating activation. Our data provide a basis for understanding the changes that REC and output domains undergo to set a default "inactive" state.

A comprehensive understanding of the molecular mechanisms underpinning cellular functions is dependent on a detailed characterization of the energetics of macromolecular binding, often quantified by the equilibrium dissociation constant, KD. While many biophysical methods may be used to obtain KD, the focus of this report is a relatively new method called “microscale thermophoresis” (MST). In an MST experiment, a capillary tube filled with a solution containing a dye-labeled solute is illuminated with an infrared laser, rapidly creating a temperature gradient. Molecules will migrate along this gradient, causing changes in the observed fluorescence. Because the net migration of the labeled molecules will depend on their liganded state, a binding curve can be constructed as a function of ligand concentration from MST data and analyzed to determine KD. Herein, simulations demonstrate the limits of KD that can be measured in current instrumentation. They also show that binding kinetics are a major concern when planning and executing MST experiments. Additionally, studies of two protein-protein interactions illustrate challenges encountered in acquiring and analyzing MST data. Combined, these approaches indicate a set of best practices for performing and analyzing MST experiments. Software for rigorous data analysis is also introduced.

Metallic residues are distributed heterogeneously onto small-arms range soils from projectile fragmentation upon impact with a target or berm backstop. Incremental Sampling Methodology (ISM) can address the spatially heterogeneous contamination of surface soils on small-arms ranges, but representative kilogram-sized ISM subsamples are affected by the range of metallic residue particle sizes in the sample. This study compares the precision and concentrations of metals in a small-arms range soil sample processed by a puck mill, ring and puck mill, ball mill, and mortar and pestle prior to analysis. The ball mill, puck mill, and puck and ring mill produced acceptable relative standard deviations of less than 15% for the anthropogenic metals of interest (Lead (Pb), Antimony (Sb), Copper (Cu), and Zinc (Zn)), with the ball mill exhibiting the greatest precision for Pb, Cu, and Zn. Precision by mortar and pestle, without milling, was considerably higher (40% to >100%) for anthropogenic metals. Median anthropogenic metal concentrations varied by more than 40% between milling methods, with the greatest concentrations produced by the puck mill, followed by the puck and ring mill and then the ball mill. Metal concentrations were also dependent on milling time, with concentrations stabilizing for the puck mill by 300 s but still increasing for the ball mill over 20 h. Differences in metal concentrations were not directly related to the surface area of the milled sample. Overall, the tested milling methods were successful in producing reproducible data for soils containing metallic residues. However, the effects of milling type and time on concentrations require consideration in environmental investigations.

Significance We assess the trade-offs and synergies involved with coordinated dam removal at three spatial scales in New England. We find that increasing the scale of dam decisions improves trade-offs among ecosystem services, river safety, and cost, but the benefits of large-scale river restoration vary dramatically by location. Our model may help facilitate future dam decision negotiations by identifying appropriate scales, locations, and criteria that satisfy multilateral funding, policy, and stakeholder goals., Aging infrastructure and growing interests in river restoration have led to a substantial rise in dam removals in the United States. However, the decision to remove a dam involves many complex trade-offs. The benefits of dam removal for hazard reduction and ecological restoration are potentially offset by the loss of hydroelectricity production, water supply, and other important services. We use a multiobjective approach to examine a wide array of trade-offs and synergies involved with strategic dam removal at three spatial scales in New England. We find that increasing the scale of decision-making improves the efficiency of trade-offs among ecosystem services, river safety, and economic costs resulting from dam removal, but this may lead to heterogeneous and less equitable local-scale outcomes. Our model may help facilitate multilateral funding, policy, and stakeholder agreements by analyzing the trade-offs of coordinated dam decisions, including net benefit alternatives to dam removal, at scales that satisfy these agreements.

Significance Light–oxygen–voltage (LOV) domain photoreceptors are found ubiquitously in nature and possess highly diverse signaling roles and mechanisms. Here, we show that a class of fungal LOV proteins dynamically associates with anionic plasma membrane phospholipids by a blue light-switched electrostatic interaction. This reversible association is rapidly triggered by blue light and ceases within seconds when illumination ceases. Within the native host, we predict that these proteins regulate G-protein signaling by the controlled recruitment of fused regulator of G-protein signaling (RGS) domains; in applied contexts, we anticipate that engineered chimeric versions of such proteins will be useful for rapid optogenetic membrane localization of fused proteins through direct interaction with the membrane itself, without requiring additional components to direct subcellular localization., We report natural light–oxygen–voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10−7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure–function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.

Different economic development strategies may result in varied socioeconomic and environmental synergies or tradeoffs, suggesting an opportunity for environmentally conscious planning. To understand such synergies or tradeoffs, a dynamic environmental life cycle assessment was conducted for eleven groups of New Hampshire industries. Historical state level Gross Domestic Product (GDP)-by-industry data was combined with economic input-output analysis to calculate the direct and life cycle energy use, freshwater use, greenhouse gas emissions, and eutrophication potential of each industry on a yearly basis for the period of 1997–2012. The future development of agriculture, traditional manufacturing, high tech, and tourism industries were investigated based on government projections. Total life cycle impacts of the 11 industries were found to represent around three to seven times those of direct impacts, indicating the significance of the supply chain impacts. Traditional manufacturing has the highest life cycle impacts even though it contributes to less than 10% of the state GDP. Future development of high tech was found to be the best strategy to increase GDP while imposing the least additional environmental impacts. Tourism presents relatively high impacts in terms of freshwater use and eutrophication potential, and a change in recreational style might be able to reduce its impacts.

Melanoma in situ (MIS) diagnosed from a subtotal biopsy may be upstaged to invasive melanoma after resection. The frequency of this phenomenon is markedly variable.To quantify the rate of upstaging MIS on the head and neck after resection at this institution, characterize the location of the invasive component relative to the clinically evident lesion, and determine the rate of upstaging with time.The authors retrospectively reviewed clinical records of adult patients with a preoperative diagnosis of MIS on the head and neck from January 1994 to August 2012. Patient and tumor characteristics were recorded.In total, 624 patients met the inclusion criteria and 24 (4%) were upstaged after resection. Four patients had invasive disease beyond the clinically evident lesion. The annual percentage of upstaged lesions seemed to show an increasing trend with time.Upstaging of MIS on the head and neck occurs at a relatively low rate that may be increasing with time. Invasive components of lentigo maligna melanoma may exist beyond the clinically evident margins. Histological examination of the maximal amount of the surgical specimen is paramount for optimal staging and treatment of MIS.

Hypoxia inducible factor (HIF) transcription factors reside at the center of signaling pathways used by mammalian cells to sense and respond to low oxygen levels. While essential to maintain oxygen homeostasis, misregulation of HIF protein activity correlates with tumor development and metastasis. To provide artificial routes to target misregulated HIF activity, we identified small molecule antagonists of the HIF-2 transcription factor that bind an internal cavity within the C-terminal PAS domain of the HIF-2α subunit. Here we describe a new class of chiral small molecule ligands that provide the highest affinity binding, the most effective, isoform-selective inhibition of HIF-2 in cells, and trigger the largest protein conformation changes reported to date. The current results further illuminate the molecular mechanism of HIF-2 antagonism and suggest additional routes to develop higher affinity and potency HIF-2 antagonists.

To survive and adapt to environmental changes, bacteria commonly use two-component signaling systems. Minimally, these pathways use histidine kinases (HKs) to detect environmental signals, harnessing these to control phosphorylation levels of receiver (REC) domains of downstream response regulators that convert this signal into physiological responses. Studies of several prototypical REC domains suggest that phosphorylation shifts these proteins between inactive and active structures that are globally similar and well-folded. However, it is unclear how globally these findings hold within REC domains in general, particularly when they are considered within full-length proteins. Here, we present EL_LovR, a full-length REC-only protein that is phosphorylated in response to blue light in the marine α-proteobacterium, Erythrobacter litoralis HTCC2594. Notably, EL_LovR is similar to comparable REC-only proteins used in bacterial general stress responses, where genetic evidence suggests that their potent phosphatase activity is important to shut off such systems. Size exclusion chromatography, light scattering, and solution NMR experiments show that EL_LovR is monomeric and unfolded in solution under conditions routinely used for other REC structure determinations. Addition of Mg(2+) and phosphorylation induce progressively greater degrees of tertiary structure stabilization, with the solution structure of the fully activated EL_LovR adopting the canonical receiver domain fold. Parallel functional assays show that EL_LovR has a fast dephosphorylation rate, consistent with its proposed function as a phosphate sink that depletes the HK phosphoryl group, promoting the phosphatase activity of this enzyme. Our findings demonstrate that EL_LovR undergoes substantial ligand-dependent conformational changes that have not been reported for other RRs, expanding the scope of conformational changes and regulation used by REC domains, critical components of bacterial signaling systems.

Research shows grab sampling is inadequate for evaluating military ranges contaminated with energetics because of their highly heterogeneous distribution. Similar studies assessing the heterogeneous distribution of metals at small-arms ranges (SAR) are lacking. To address this we evaluated whether grab sampling provides appropriate data for performing risk analysis at metal-contaminated SARs characterized with 30–48 grab samples. We evaluated the extractable metal content of Cu, Pb, Sb, and Zn of the field data using a Monte Carlo random resampling with replacement (bootstrapping) simulation approach. Results indicate the 95% confidence interval of the mean for Pb (432 mg/kg) at one site was 200–700 mg/kg with a data range of 5–4500 mg/kg. Considering the U.S. Environmental Protection Agency screening level for lead is 400 mg/kg, the necessity of cleanup at this site is unclear. Resampling based on populations of 7 and 15 samples, a sample size more realistic for the area yielded high false negative rates.