Botulinum neurotoxin B is a Food and Drug Administration-approved therapeutic toxin. However, it has lower binding affinity toward the human version of its major receptor, synaptotagmin II (h-Syt ...II), compared to mouse Syt II, because of a residue difference. Increasing the binding affinity to h-Syt II may improve botulinum neurotoxin B's therapeutic efficacy and reduce adverse effects. Here we utilized the bacterial adenylate cyclase two-hybrid method and carried out a saturation mutagenesis screen in the Syt II-binding pocket of botulinum neurotoxin B. The screen identifies E1191 as a key residue: replacing it with M/C/V/Q enhances botulinum neurotoxin B binding to human synaptotagmin II. Adding S1199Y/W or W1178Q as a secondary mutation further increases binding affinity. Mutant botulinum neurotoxin B containing E1191M/S1199Y exhibits ~11-fold higher efficacy in blocking neurotransmission than wild-type botulinum neurotoxin B in neurons expressing human synaptotagmin II, demonstrating that enhancing receptor binding increases the overall efficacy at functional levels. The engineered botulinum neurotoxin B provides a platform to develop therapeutic toxins with improved efficacy.Humans are less sensitive to the therapeutic effects of botulinum neurotoxin B (BoNT/B) than the animal models it is tested on due to differences between the human and the mouse receptors. Here, the authors engineer BoNT/B to improve its affinity to human receptors and enhance its therapeutic efficacy.
We ask how environmental justice and urban ecology have influenced one another over the past 25 years in the context of the US Long-Term Ecological Research (LTER) program and Baltimore Ecosystem ...Study (BES) project. BES began after environmental justice emerged through activism and scholarship in the 1980s but spans a period of increasing awareness among ecologists and environmental practitioners. The work in Baltimore provides a detailed example of how ecological research has been affected by a growing understanding of environmental justice. The shift shows how unjust environmental outcomes emerge and are reinforced over time by systemic discrimination and exclusion. We do not comprehensively review the literature on environmental justice in urban ecology but do present four brief cases from the Caribbean, Africa, and Asia, to illustrate the global relevance of the topic. The example cases demonstrate the necessity for continuous engagement with communities in addressing environmental problem solving.
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Carbonic anhydrases (CAs) are metallo-enzymes that catalyze the reversible hydration of carbon dioxide into bicarbonate and a proton. The β-class CAs (β-CAs) are expressed in ...prokaryotes, fungi, plants, and more recently have been isolated in some animals. The β-CA class is divided into two subclasses, termed type I and II, defined by pH catalytic activity profile and active site structural configuration. Type I β-CAs display catalytic activity over a broad pH range (6.5–9.0) with the active site zinc tetrahedrally coordinated by three amino acids and a hydroxide/water. In contrast, type II β-CAs are catalytically active only at a pH 8 and higher where they adopt a functional active site configuration like that of type I. However, below pH 8 they are conformationally self-inactivated by the addition of a fourth amino acid coordinating the zinc and thereby displacing the zinc bound solvent. We have determined the structure of psCA3, a type II β-CA, isolated from Pseudomonas aeruginosa (P. aeruginosa) PAO1 at pH 8.3, in its open active state to a resolution of 1.9Å. The active site zinc is coordinated by Cys42, His98, Cys101 and a water/hydroxide molecule. P. aeruginosa is a multi-drug resistant bacterium and displays intrinsic resistance to most of the currently used antibiotics; therefore, there is a need for new antibacterial targets. Kinetic data confirm that psCA3 belongs to the type II subclass and that sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid are micromolar inhibitors. In vivo studies identified that among six tested inhibitors representing sulfonamides, inorganic anions, and small molecules, acetazolamide has the most significant dose-dependent inhibitory effect on P. aeruginosa growth.
As national economies continue to evolve across the globe, businesses are increasing their capacity to not only generate new products and deliver them to customers, but also to increase levels of ...after-sales service. One major component of after-sale service involves service parts management. However, service parts businesses are typically seen as add-ons to existing business models, and are not well integrated with primary businesses. Consequently, many service parts operations are managed using ad-hoc practices that are often subordinated to primary businesses. Early research in this area has been instrumental in assisting organizations to begin optimizing some aspects of service parts management. However, performance goals for service parts management are often ill-defined. Further, because these service parts businesses are often subordinated to primary businesses within a firm, the use of newer big data applications to help manage these processes is almost completely absent. Herein, we develop a framework that seeks to define service parts performance goals for the purpose of outlining where scholars and practitioners can further examine where, how, and why big data applications can be employed to enhance service parts management performance.
Monocarboxylate transporters (MCTs) mediate the proton-coupled exchange of high-energy metabolites, including lactate and pyruvate, between cells and tissues. The transport activity of MCT1, MCT2, ...and MCT4 can be facilitated by the extracellular carbonic anhydrase IV (CAIV) via a noncatalytic mechanism. Combining physiological measurements in HEK-293 cells and Xenopus oocytes with pulldown experiments, we analyzed the direct interaction between CAIV and the two MCT chaperones basigin (CD147) and embigin (GP70). Our results show that facilitation of MCT transport activity requires direct binding of CAIV to the transporters chaperones. We found that this binding is mediated by the highly conserved His-88 residue in CAIV, which is also the central residue of the enzyme’s intramolecular proton shuttle, and a charged amino acid residue in the Ig1 domain of the chaperone. Although the position of the CAIV-binding site in the chaperone was conserved, the amino acid residue itself varied among different species. In human CD147, binding of CAIV was mediated by the negatively charged Glu-73 and in rat CD147 by the positively charged Lys-73. In rat GP70, we identified the positively charged Arg-130 as the binding site. Further analysis of the CAIV-binding site revealed that the His-88 in CAIV can either act as H donor or H acceptor for the hydrogen bond, depending on the charge of the binding residue in the chaperone. Our results suggest that the CAIV-mediated increase in MCT transport activity requires direct binding between CAIV–His-88 and a charged amino acid in the extracellular domain of the transporter’s chaperone.
•Modern political economy can frame urban transitions to sustainability and global environmental change.•The choice of urban institutions for urban transitions requires a comprehensive assessment of ...uncertainties inherent to decision-making.•Transitions can be complicated by self-referential adaptation and reformation mechanisms inherent in institutions.•Good urban governance depends on the capacities of a nexus of actors as well as their willingness to engage in participatory processes.
Our paper has two major aims: to argue that lessons from modern political economy are critical for urban transitions to sustainability and to position relevant urban sustainability transitions literature within distinct focus areas of modern political economy—in particular, endogenous institutions. We address those aims by providing examples of recent scholarship on institutional change and modern urban political economy that has significant implications for the understanding of urban transitions to sustainability. Furthermore, we explore the relevance of endogenous institutions for global environmental change and urban sustainability transitions; we utilize three relevant conceptual areas: (i) the choice of institutions and institutional change; (ii) the maintenance of institutions and institutional robustness; (iii) effective or ‘good’ urban governance and government failures.
Proton-coupled monocarboxylate transporters (MCTs) mediate the exchange of high energy metabolites like lactate between different cells and tissues. We have reported previously that carbonic ...anhydrase II augments transport activity of MCT1 and MCT4 by a noncatalytic mechanism, while leaving transport activity of MCT2 unaltered. In the present study, we combined electrophysiological measurements in Xenopus oocytes and pulldown experiments to analyze the direct interaction between carbonic anhydrase II (CAII) and MCT1, MCT2, and MCT4, respectively. Transport activity of MCT2-WT, which lacks a putative CAII-binding site, is not augmented by CAII. However, introduction of a CAII-binding site into the C terminus of MCT2 resulted in CAII-mediated facilitation of MCT2 transport activity. Interestingly, introduction of three glutamic acid residues alone was not sufficient to establish a direct interaction between MCT2 and CAII, but the cluster had to be arranged in a fashion that allowed access to the binding moiety in CAII. We further demonstrate that functional interaction between MCT4 and CAII requires direct binding of the enzyme to the acidic cluster 431EEE in the C terminus of MCT4 in a similar fashion as previously shown for binding of CAII to the cluster 489EEE in the C terminus of MCT1. In CAII, binding to MCT1 and MCT4 is mediated by a histidine residue at position 64. Taken together, our results suggest that facilitation of MCT transport activity by CAII requires direct binding between histidine 64 in CAII and a cluster of glutamic acid residues in the C terminus of the transporter that has to be positioned in surroundings that allow access to CAII.
Background: Carbonic anhydrase II (CAII) augments activity of monocarboxylate transporters (MCTs) by noncatalytic interaction.
Results: CAII binds to an acidic cluster with an appropriate context in the MCT C terminus.
Conclusion: Isoform-specific interaction between MCTs and CAII requires a specific binding moiety.
Significance: CAII-mediated increase in lactate transport depends on the presence of a specific binding moiety in MCT.
The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration of CO2 to bicarbonate and a proton. Recently, there has been industrial interest in ...utilizing CAs as biocatalysts for carbon sequestration and biofuel production. The conditions used in these processes, however, result in high temperatures and acidic pH. This unfavorable environment results in rapid destabilization and loss of catalytic activity in CAs, ultimately resulting in cost‐inefficient high‐maintenance operation of the system. In order to negate these detrimental industrial conditions, cysteines at residues 23 (Ala23Cys) and 203 (Leu203Cys) were engineered into a wild‐type variant of human CA II (HCAII) containing the mutation Cys206Ser. The X‐ray crystallographic structure of the disulfide‐containing HCAII (dsHCAII) was solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active‐site geometry. Kinetic studies utilizing the measurement of 18O‐labeled CO2 by mass spectrometry revealed that dsHCAII retained high catalytic efficiency, and differential scanning calorimetry showed acid stability and thermal stability that was enhanced by up to 14 K compared with native HCAII. Together, these studies have shown that dsHCAII has properties that could be used in an industrial setting to help to lower costs and improve the overall reaction efficiency.
The Earth’s population will become more than 80% urban during this century. This threshold is often regarded as sufficient justification for pursuing urban ecology. However, pursuit has primarily ...focused on building empirical richness, and urban ecology theory is rarely discussed. The Baltimore Ecosystem Study (BES) has been grounded in theory since its inception and its two decades of data collection have stimulated progress toward comprehensive urban theory. Emerging urban ecology theory integrates biology, physical sciences, social sciences, and urban design, probes interdisciplinary frontiers while being founded on textbook disciplinary theories, and accommodates surprising empirical results. Theoretical growth in urban ecology has relied on refined frameworks, increased disciplinary scope, and longevity of interdisciplinary interactions. We describe the theories used by BES initially, and trace ongoing theoretical development that increasingly reflects the hybrid biological–physical–social nature of the Baltimore ecosystem. The specific mix of theories used in Baltimore likely will require modification when applied to other urban areas, but the developmental process, and the key results, will continue to benefit other urban social–ecological research projects.
As highly managed ecosystems, urban areas should reflect the social characteristics of their managers, who are primarily residents. Since landscape features develop over time, we hypothesize that ...present-day vegetation should also reflect social characteristics of past residents. Using an urban-to-suburban watershed in the Baltimore Metropolitan Region, this paper examines the relationship between demographics, housing characteristics, and lifestyle clusters from 1960 and 2000 with areas of high woody and herbaceous vegetation cover in 1999. We find that 1960 demographics and age of housing are better predictors of high woody or tree coverage in 1999 than demographics and housing characteristics from 2000. Key variables from 1960 are percent in professional occupations (+), percent of pre-WWI housing (−), percent of post-WWII housing (+), and population density (−). Past and present demographic and housing variables are poor predictors of high herbaceous cover in 1999. Lifestyle clusters for 2000 are very good predictors of high herbaceous coverage in 1999, but lifestyle clusters from 1960 and 2000 are poor predictors of high woody vegetation coverage. These findings suggest that herbaceous or grassy areas, typically lawns, are good reflections of contemporary lifestyle characteristics of residents while neighborhoods with heavy tree canopies have largely inherited the preferred landscapes of past residents and communities. Biological growth time scales of trees and woody vegetation means that such vegetation may outlast the original inhabitants who designed, purchased, and planted them. The landscapes we see today are therefore legacies of past consumption patterns.
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