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Supplementary Figure 1 from Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity

The identification of small molecules that inhibit the sequence-specific binding of transcription factors to DNA is an attractive approach for regulation of gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that controls genes involved in glycolysis, angiogenesis, migration, and invasion, all of which are important for tumor progression and metastasis. To identify inhibitors of HIF-1 DNA-binding activity, we expressed truncated HIF-1α and HIF-1β proteins containing the basic-helix-loop-helix and PAS domains. Expressed recombinant HIF-1α and HIF-1β proteins induced a specific DNA-binding activity to a double-stranded oligonucleotide containing a canonical hypoxia-responsive element (HRE). One hundred twenty-eight compounds previously identified in a HIF-1–targeted cell-based high-throughput screen of the National Cancer Institute 140,000 small-molecule library were tested in a 96-well plate ELISA for inhibition of HIF-1 DNA-binding activity. One of the most potent compounds identified, echinomycin (NSC-13502), a small-molecule known to bind DNA in a sequence-specific fashion, was further investigated. Electrophoretic mobility shift assay experiments showed that NSC-13502 inhibited binding of HIF-1α and HIF-1β proteins to a HRE sequence but not binding of the corresponding proteins to activator protein-1 (AP-1) or nuclear factor-κB (NF-κB) consensus sequences. Interestingly, chromatin immunoprecipitation experiments showed that NSC-13502 specifically inhibited binding of HIF-1 to the HRE sequence contained in the vascular endothelial growth factor (VEGF) promoter but not binding of AP-1 or NF-κB to promoter regions of corresponding target genes. Accordingly, NSC-13502 inhibited hypoxic induction of luciferase in U251-HRE cells and VEGF mRNA expression in U251 cells. Our results indicate that it is possible to identify small molecules that inhibit HIF-1 DNA binding to endogenous promoters.

Supplementary Figure 2 from Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity

In recent decades, out-migration has become a key livelihood strategy for many rural households in the middle hills region of Nepal. In this region, rural communities are key actors in the management of local resources such as community forests. Analysis of the link between community forests and out-migration is largely missing in the literature, even though the demographic changes associated with out-migration affect forest resource use and management. This article discusses how rural communities and traditional landscapes in the middle hills are changing following out-migration and how this process has changed the management by rural communities of local forests (including community forests). Overall, the research found a reduced dependency by households on forest products (e.g. firewood, fodder and timber) sourced from community forests. Also, the reduced demand is being supplied increasingly from trees/forests grown on private farmland, including natural regenerated forests and trees planted on abandoned farmland. The declining need for forest products and the lack of an economic incentive for active forest management coupled with a decreasing sense of community has reduced the interest in community forests, leading to less intensive and infrequent forest management. Given the prevalence of out-migration and the changing socio-economic context in the middle hills, it appears time to reconsider the management of community forests beyond a narrow range of uses to enable greater commercialization and encourage ecosystem services to be harnessed so community forests better align with contemporary rural livelihoods and landscapes.

We offer a unified conceptual, behavioral, and econometric framework for optimal fundraising that deals with both synergies and discrepancies between approaches from Economics, Marketing, Psychology, and Sociology. The purpose is to offer a framework that can bridge differences and open a dialogue between disciplines in order to facilitate optimal fundraising design. The literature is extensive, and our purpose is to offer a brief background and perspective on each of the approaches, provide an integrated framework leading to new insights, and discuss areas of future research.

Should advertisers change their message (what is said), just as they do the execution (how it is said) to reflect changing consumer preferences? This paper is the first to quantify how these ad components and their interplay affect brand sales. The authors define the concepts of Market Consistency and changes in ad executions and show how they interact with each other and with the brand’s age in their sales outcome. The empirical analysis confirms the hypotheses in the U.S. minivan market. As a brand matures, executional variations become increasingly beneficial, but changing advertising messaging to remain consistent with customer preferences becomes less effective. For older brands with little executional variation, changing the ad message even reduces sales.The authors thus uncover important boundary conditions for the opposing theories that brands should ‘stick with their message’ versus ‘change with the times’ and advice how to manage advertising as the brand matures.

Environmental and development discourses in China can be categorised into three narrative motifs framing human–nature relationships: peasant, indigenous, and community. Indigenous and community narratives have been widely adopted by environmental NGOs (eNGOs) in China in promoting community-based natural resource management projects, but there has been very limited critical research on such phenomena. Analysis of socio-economic change in two ethnic minority communities in Yunnan shows that neither narrative theme is fully internalised by the relevant communities. Instead narratives may be strategically modified or even rejected by local communities. This is due to different agendas being held by local communities and eNGOs, and two factors pertinent to rural China: the incompatibility of concepts of ‘community’ in Chinese and international contexts results in confusion, and a lack of recent territorial and cultural claims by rural communities since the collectivist era makes it difficult to construct the identity of a community. It remains challenging for eNGOs in China to advocate either community or indigenous narratives in contexts of rapid socio-economic change.

Continuous manufacturing (CM) is an emerging technology in the pharmaceutical manufacturing sector, and the understanding of the impact on product quality is currently evolving. As the final purification and isolation step, crystallization has a significant impact on the final physicochemical properties of drug substance and is considered a critical process step in achieving the continuous manufacturing of drug substances. Although many publications previously focused on various innovative techniques to continuously make crystals with desired properties, engineering difficulties such as system design, automation, and integration with process analytical technology (PAT) tools have not been thoroughly discussed. Here, we focus on how to develop a continuous crystallization system, from the perspective of process engineering, and the related risk considerations on product quality. Specifically, we designed and built an automated two-stage mixed suspension mixed product removal (MSMPR) crystallization platfor...

For continuous manufacturing of pharmaceuticals, understanding the dynamics of how a material flows through the process is critical with respect to the development of a control strategy for product quality assurance. Such understanding of the process dynamics can be obtained by characterization of the residence time distribution (RTD). The RTD for a process is not fixed and can vary due to changes in operating conditions or physiochemical properties of the blend. As such the RTD needs to be evaluated over the range of operating condition that can impact process dynamics (e.g. throughput, impeller rotation rate etc.). In this paper, we demonstrate that the dimensionless RTD (normalized with respect to the mean residence time) is invariant with throughput and impeller rotation rates under certain conditions for the two continuous direct compression processes. We present a case study to illustrate the utility of this relationship for predicting the process dynamics at different operating conditions (i.e., throughputs) and evaluating the impact of variations in the process dynamics on the control strategy for a continuous direct compression process.

Given that consumers value quality, and advertising content informs consumers’ beliefs about quality, it is not surprising that high-quality brands emphasize quality in their advertising content. What is less obvious is whether firms with lower-quality brands should also follow suit and emphasize quality in their advertising to signal a higher quality. We examine this issue and study the effectiveness of quality-based advertising messages. Our field study relates brands’ monthly sales to their advertised quality claims across 1,876 print ads in national magazines and Consumer Reports–based product quality ratings over more than two decades. Contrary to the generally held yet erroneous belief in the efficacy of low-quality products emphasizing quality in their advertising, we demonstrate that (1) it is not beneficial for a low-quality firm to emphasize quality in its advertising, and (2) it is effective for a high-quality firm to do so. An analysis of parameter values from a published category-agnostic simulation and an experiment that examines consumers’ responses to quality claims in a second product category yields convergent insights.

Forest and landscape restoration are emerging globally as a major challenge for development and conservation in the twenty-first century. With a restoration market providing demand for participation in supplying products and services, such as native seed, Indigenous communities have experienced new opportunities for cash income and livelihood improvements. This chapter explores the ways different Indigenous populations in Brazil have engaged in the native seed trade for restoring degraded lands, and the outcomes of their participation. We cover a case study based on one decade’s experience of the Xingu Seeds Network, focusing on challenges faced by 232 Indigenous people, mostly women, from 13 villages of Ikpeng, Kawaiwete, Matipu, Panara, Wauja, Xavante and Yudja ethnicity in the southeastern Amazon. Indigenous communities have engaged in the activity mainly to secure current and future access to natural resources. Although communities have shown a large fluctuation in their seed production and cash income over the years, these collectors have produced 6.7 tonnes of seeds from 159 species leading to US$ 65 thousand cash income for households. Native seed production is strongly aligned with traditional knowledge with outcomes related to women’s empowerment, opportunities for young people, territorial mapping, and building local organizations. However, communities have faced constraints in adapting ethnic institutions to business management requirements mainly due to scales of production, language difficulties and lack of accounting skills. Therefore, institutions must be developed acknowledging Indigenous knowledge and culture for building a more inclusive and flexible approach to support Indigenous groups to adapt to participation in markets.

The official approach to urban planning in Thailand involves a centralized process that relies mainly on decisions from the national government. This approach has not been effective in a small and unique area like Wat Ket in Chiang Mai City in northern Thailand which has a variety of different land use types. Some land use areas in the Wat Ket area were designated as commercial zones, without the involvement and agreement of local residents and this led to land use conflicts and other related problems. This paper describes how stakeholders in Wat Ket applied an alternative collaborative approach to urban land use planning. The paper provides a case study of the collaborative urban planning approach in the context of a rapidly growing city in the developing world.Collaborative urban land use planning is supported by land use analysis applying aerial photographs and Geographical Information Systems (GIS) to analyse land use changes in the study area.

Large-scale ecological restoration programs across the world involve a voluminous demand for native seeds of diverse native plant species. In this article, we explore how institutional systems have operated and impacted native seed supply in Brazil. Native seed supply for restoration is essentially a community-based activity which faces broad barriers to operating within regulations because of requirements for excessive and costly technical documentation, scarcity of seed laboratories, and lack of instructions for native seed quality testing. Although decentralized seed networks have stimulated arrangements for local organizations to promote seed supply, policies constrain the development of local capacities and the ongoing sustainability of these organizations. These conditions have resulted in a vast network of informal collectors and producers who are largely “invisible” and unknown to the regulatory authorities. Policies have decentralized responsibilities from the state without devolving decision-making power to the multiple stakeholders engaged in policy elaboration. The policies maintain the centralized regulation of native seed supply. After examining Brazilian seed networks’ experiences and conducting discussions with stakeholders and experts, we suggest adapting the current regulations to more local level contexts, encompassing the following strategies: (1) ensuring native seed origin and identity; (2) relaxation of the laboratory accreditation process for native seed quality assurance; (3) fostering seed markets for restoration; (4) research to provide technological innovation; (5) supporting local, diverse, and small seed-based businesses.

The utility of shell overlays to oyster (Crassostrea virginica) plantings as a cownose ray (Rhinoptera bonasus) predator deterrence mechanism was examined. Typical industry practice of oyster seed planting was followed in an experimental design employing treatment areas of 0.5–1.0 acre (0.2–0.4 hectare). Areas were prepared in the Lower Machodoc Creek, Virginia, by the initial application of shell to insure a stable substrate under planted seed oysters. Seed oysters were planted using standard industry methods. Experimental areas were located, two upstream and two downstream, of a constriction in the Lower Machodoc that dictated differing physical environments in the respective locations with downstream locations being more exposed to northeast wind-driven stresses and, historically, a greater incidence of ray predation. Once oysters were planted, two of the areas, one upstream and one downstream of the aforementioned constriction, were additionally treated with a shell overlay as a predation det...

There is increased research into pharmaceutical nanoemulsions where the timing of quantification of active components can dictate continuous manufacturing production cost and consistency. The goal of this study was development of a rapid quantification method for isoflurane nanoemulsions using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR). Isoflurane was quantified by ATR-FTIR and High-Performance Liquid Chromatography (HPLC). Correlation and agreement between methods was determined to validate the ATR-FTIR procedure. Evaporation rate studies from the nanoemulsions and pure isoflurane were performed. ATR-FTIR values were in agreement and correlated with (HPLC) measurements (calculated Pearson R of 0.99; 99 % confidence interval; P

Over the last few decades, numerous initiatives have advanced forest landscape restoration in the Amazon, and in 2015 the Brazilian government set an ambitious, still-valid, target to restore 4.8 million hectares of degraded Amazonian land by 2030. This has contributed to an emergent global restoration network that connects multiple stakeholders and processes for funding, implementing and monitoring restoration actions in such a way that prepares various ecosystem services for market integration. The network arose in tandem with the evolution of an institutional framework that includes regulatory requirements within Brazil, global commitments linked to climate change mitigation, corporate sustainability strategies, and the growth of crowd-sourcing activism. This paper presents restoration activities as embedded within a Global Production Network (GPN) for an ecosystem service, which we use as a heuristic device to inform our understanding of emergent environmental governance structures. The resulting multi-scalar, networked mode of environmental governance is presented as a web-like structure co-created by institutional evolution, actor-specific strategies, and interactions between firms and non-firm actors. The article pays particular attention to a case study of how the restoration network manifests territorially in the Upper Xingu region of the Brazilian Amazon. Despite the strong North-South orientation of dominant funding relationships, network governance is also seen to be relational. This is evident from the dissemination of ideas, supply models and seeding techniques from Upper Xingu to other regions of Brazil. These insights could be applied to improve landscape restoration outcomes, and indeed the provisioning of ecosystem services more broadly.

In this paper, we explore how diverse community networks in Brazil have locally advanced seed production and institutional systems to enhance a restoration economy. By focusing on the experiences of the six major native seed suppliers in Amazonia, the Cerrado, and the Atlantic Forest, we estimate the capacity to scale-up community-based systems to meet a large-scale restoration target as a rural development strategy. Over one decade, 1016 collectors traded 416.91 tonnes of native seeds representing, on average, 31.41 kilos yearly and USD 256.5 as household income. Based on this well documented empirical evidence, we estimate that Brazil&rsquo, s restoration goal would require from 3.6 to 15.6 thousand tonnes of native seeds depending on the share of each restoration method adopted with potential work opportunities for 13.2 to 57.1 thousand collectors yearly and total income from USD 34 to 146 million. We argue that community networks represent feasible arrangements for increasing the availability of plant material sources which provide high socio-economic benefits. For scaling up native seed sources, we suggest the following key strategies: (i) government incentives and subsidies, (ii) enforcement of ecosystem restoration, (iii) community participation, (iv) adaptation of the seed regulations, (v) technological development, and (vi) seed market diversification.

While community forestry has shown promise to reduce rural poverty, improve reforestation and potentially offset carbon emissions, many projects have failed, either partly or completely. In order to understand why community forestry succeeds or fails, we examined in detail the literature related to community forestry from three countries, Mexico, Nepal and the Philippines. We also drew on experiences in other countries in Asia, Latin America and Africa. We identified five main interconnected factors which the literature suggests are often critical to the success of community forestry. To integrate the many ways in which community forestry projects can improve the state of these factors, we use the concept of ‘bonding social capital’, i.e. communities’ ability to work together towards a common aim and ‘bridging social capital’, i.e. their ability to liaise with the outside world. To understand the interaction of the five success factors and the way in which improvements to bonding or bridging social capital may affect them, we developed a causal diagram which depicts the interrelationships between the success factors and the key points at which project inputs may be best applied. It is clear from our analysis that failing to appreciate both the complexity and interaction of the various influences may lead to project failure.

Community forestry focuses on the link between forest resources and livelihoods and contributes to forest conservation and reforestation. It is widespread in Nepal, with a very high proportion of the rural population involved, and is widely recognized as one of the most successful examples of community forestry in Asia. Through a combination of literature reviews and original research, this volume explores key experiences and outcomes of community forestry in Nepal over the last four decades as a model for improving forest management and supporting local livelihoods. The book takes a critical approach, recognizing successes, especially in forest conservation and restoration, along with mixed outcomes in terms of poverty reduction and benefits to forest users. It recognizes the way that community forestry has continued to evolve to meet new challenges, including the global challenges of climate change, environmental degradation and conservation, as well as national demographic and social changes due to large-scale labour migration and the growing remittance economy. In addition to examining the changes and responses, the book explores ways that community forestry in Nepal might move forward. Lessons from Nepal have relevance to community forestry and community-based approaches to natural resource management around the world that are also experiencing global pressures and opportunities. [Book Synopsis]

Community forestry focuses on the link between forest resources and livelihoods and contributes to forest conservation and reforestation. It is widespread in Nepal, with a very high proportion of the rural population involved, and is widely recognized as one of the most successful examples of community forestry in Asia. Through a combination of literature reviews and original research, this volume explores key experiences and outcomes of community forestry in Nepal over the last four decades as a model for improving forest management and supporting local livelihoods. The book takes a critical approach, recognizing successes, especially in forest conservation and restoration, along with mixed outcomes in terms of poverty reduction and benefits to forest users. It recognizes the way that community forestry has continued to evolve to meet new challenges, including the global challenges of climate change, environmental degradation and conservation, as well as national demographic and social changes due to large-scale labour migration and the growing remittance economy. In addition to examining the changes and responses, the book explores ways that community forestry in Nepal might move forward. Lessons from Nepal have relevance to community forestry and community-based approaches to natural resource management around the world that are also experiencing global pressures and opportunities. [Book Synopsis]

The research examines how the attractiveness of children in need affects the empathy they evoke and the subsequent help they receive from unrelated adults. The authors find that attractive children are attributed desirable characteristics related to social competence, which is consistent with the "beautiful is good" stereotype. Ironically, the authors find that these attributions reduce the empathy evoked by attractive children and the help they receive from unrelated adults as long as their need is not severe. These effects are demonstrated in four experiments. The research identifies a significant cost of being beautiful and an important exception to the beautiful is good stereotype. The results also have practical implications for how children are portrayed in promotional materials for disaster relief agencies, children's hospitals, and other charities.

Parents and other adults such as baby sitters, daycare workers, and teachers are responsible for making choices that contribute to the development and well-being of the children in their care. In a typical day, for example, parents decide what and how much their children eat, the amount of time they spend online, the television programs they watch, and the time they go to bed. In this research, we identify two common approaches -imposition and accommodation-that are available to decision makers and outline how these decisions affect the feelings of power and eating behaviors of the adults themselves. Four experiments and one field study demonstrate that adults shape their own consumption choices based on whether they accommodate the virtuous (i.e. healthy) vs. indulgent (i.e. unhealthy) preferences of the child or impose their own preferences. We find that when adults accommodate the child's preferences, they choose healthier foods for themselves. Finally, we demonstrate that the social context of consumption moderates these effects. We find support for our power-based theory in five studies using a variety of methods, participants, and contexts. The results have important implications for the health of adults who take care of children.

The described oxime-based library protocol provides detailed procedures for the linkage of aminooxy functionality with aldehyde building blocks that result in the generation of libraries of multidentate inhibitors. Synthesis of inhibitors for protein tyrosine phosphatases (PTPs) and antagonists directed against the human tumor susceptibility gene 101 (TSG101) are shown as examples. Three steps are involved: (i) the design and synthesis of aminooxy platforms; (ii) tethering with aldehydes to form oxime-based linkages with sufficient purity; and (iii) direct in vitro biological evaluation of oxime products without purification. Each coupling reaction is (i) performed in capped microtubes at room temperature (20-23 °C); (ii) diluted for inhibitory evaluation; and (iii) screened with targets in microplates to provide IC(50) or K(d) values. The synthesis of the aminooxy platforms takes 3-5 d; tethering with the aldehydes takes 24 h; and inhibition assay of enzymes and protein-protein interactions takes 30 min and 2 h, respectively.

What do American adults believe about what, where, when, how much, and how often it is appropriate to eat? Such normative beliefs originate from family and friends through socialization processes, but they are also influenced by governments, educational institutions, and businesses. Norms therefore provide an important link between the social environment and individual attitudes and behaviors. This paper reports on five studies that identify, develop, and validate measures of normative beliefs about eating. In study 1 we use an inductive method to identify what American adults believe are appropriate or desirable eating behaviors. Studies 2 and 3 are used to purify and assess the discriminant and nomological validity of the proposed set of 18 unidimensional eating norms. Study 4 assesses predictive validity and finds that acting in a norm-consistent fashion is associated with lower Body Mass Index (BMI), and greater body satisfaction and subjective health. Study 5 assesses the underlying social desirability and perceived healthiness of the norms.

Since decades, varieties of amphiphilic polymers have been widely investigated for improving aqueous solubility and bioavailability of the hydrophobic drugs. The upcoming approach is to develop more efficient advanced nano-carrier molecules capable of more than drug delivery. Herein, we report the design and synthesis of some novel carrier molecules with multiple applications including drug encapsulation, drug delivery and diagnosis (imaging). Copolymers were synthesized using dimethyl 5-hydroxy/aminoisophthalate, poly(ethylene glycols) and Candida antarctica lipase (CAL-B, Novozym 435). CAL-B selectively catalyses the trans esterification reaction under solvent less condition using primary hydroxyls of poly(ethylene glycols) and leaving behind phenolic hydroxyl for post polymerization modifications. The obtained copolymers were further tethered with perfluorinated aliphatic chains to make them amphiphilic. The synthesized materials were investigated for their micellar behavior, temperature dependent stability (in aqueous solution), encapsulation capacity, and imaging potential by measuring the sensitivity of these perfluorinated materials towards 19 F NMR in NMR tube. It was observed that perfluorinated amphiphilic copolymers could encapsulate up to 14% (by wt) of hydrophobic drug and showed decent 19 F NMR signals even at a very low concentration. Therefore, these perfluorinated copolymers hold considerable potential for further investigation as advanced nano-carrier molecules for biomedical applications.

We conduct three experiments in which participants in dyads choose between two restaurants, each of which is preferred by only one participant, and one participant has the power to decide which restaurant both will patronize. We find that the power to make a joint decision increases satisfaction with the choice only when those involved have a competitive decision orientation, a weak or anonymous relationship, and the outcome they choose is subsequently available. Participants who have a cooperative orientation are satisfied whether or not they have power and whether or not the resulting choice is consistent with their initial preferences.

To date, proteomic analyses on gastrointestinal cancer tissue samples have been performed using surgical specimens only, which are obtained after a diagnosis is made. To determine if a proteomic signature obtained from endoscopic biopsy samples could be found to assist with diagnosis, frozen endoscopic biopsy samples collected from 63 gastric cancer patients and 43 healthy volunteers were analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. A statistical classification model was developed to distinguish tumor from normal tissues using half the samples and validated with the other half. A protein profile was discovered consisting of 73 signals that could classify 32 cancer and 22 normal samples in the validation set with high predictive values (positive and negative predictive values for cancer, 96.8% and 91.3%; sensitivity, 93.8%; specificity, 95.5%). Signals overexpressed in tumors were identified as alpha-defensin-1, alpha-defensin-2, calgranulin A, and calgranulin B. A protein profile was also found to distinguish pathologic stage Ia (pT1N0M0) samples (n = 10) from more advanced stage (Ib or higher) tumors (n = 48). Thus, protein profiles obtained from endoscopic biopsy samples may be useful in assisting with the diagnosis of gastric cancer and, possibly, in identifying early stage disease.

The subject of envelopes has been part of differential geometry from the beginning. This paper brings a modern perspective to the classical problem of envelopes of families of affine hyperplanes. In the process, the classical results are generalized and unified. A key step in this process is the use of “generalized immersions”. These have been described elsewhere but, briefly, every classical immersion defines a generalized immersion in a canonical way so that generalized immersions can be understood as ordinary immersions “with singularities.” Next, the concept of an envelope is given a modern definition, namely, an envelope is a generalized immersion solving the family that has a universal mapping property relative to all other full rank solutions. The beauty of this approach becomes apparent in the “Envelope Theorem”. With one mild assumption, namely that the associated family of linear hyperplanes is immersed, it is proven that a family of affine hyperplanes always has an envelope, and that envelope is essentially unique.

The human epidermal growth factor receptors, EGFR and HER2, are members of the EGFR family of cell-surface receptors/tyrosine kinases. EGFR- and HER2-positive cancers represent a more aggressive disease with greater likelihood of recurrence, poorer prognosis, and decreased survival rate, compared to EGFR- or HER2-negative cancers. The details of HER2 proto-oncogenic functions are not deeply understood, partially because of a restricted availability of tools for EGFR and HER2 detection (A. Sorkin and L. K. Goh, Exp. Cell Res. 2009, 315, 683-696). We have created photostable and relatively simple-to-produce imaging probes for in vitro staining of EGFR and HER2. These new reagents, called affiprobes, consist of a targeting moiety, a HER2- or EGFR-specific Affibody molecule, and a fluorescent moiety, mCherry (red) or EGFP (green). Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio of affiprobes. Affiprobes are able to stain both live cells and frozen tumor xenograph sections. This type of optical probe can easily be extended for targeting other cell-surface antigens/ receptors.

Viral release is a necessary component of HIV-1 replication. To be an efficient process, viral budding relies on recruitment of the ubiquitin E2 variant (UEV) domain of the human tumor susceptibility gene 101 protein (Tsg101) by major structural proteins of HIV-1.1,2 This entails the direct interaction of the Tsg101 UEV domain with a proline rich motif (PRM) within the viral Gagp6 protein that contains a conserved sequence, Pro-Thr/Ser-Ala-Pro [“P(T/S)AP”].3,4 Over-expression of the Tsg101 UEV inhibits virus release by interfering with processing of the p6 domains and this effect is abrogated by mutation within the P(T/S)AP binding site. In theory, blocking this critical Tsg101-p6 interaction could prevent viral budding and provide a basis for new targeted antiretroviral therapies.5,6 This is supported by the recent finding that inhibition of HIV budding can be achieved by cyclic peptides that interfere with Tsg101-Gag interactions.7 In an effort to develop Tsg101-binding inhibitors using the reported p6-derived 9-mer wild-type (WT) sequence “P1E2P3T4A5P6P7E8E9,” we had previously improved Tsg101-binding affinity by applying hydrazone- and hydrazide-library techniques.8 We also reported an oxime-based post solid-phase diversification approach to peptide library construction.9–11 However, the peptide mimetics resulting from this earlier work showed poor biovailability in cell-based experiments. Because poor cellular uptake was thought to contribute to the low bioavailability, peptides were conjugated with known membrane carrier antennapedia and HIV-Tat (48–60) peptides.12 Although these constructs did exhibit apparent enhanced cellular uptake, the Tsg101 binding affinity of the conjugates was seriously disrupted. Therefore, we sought an alternate approach that could improve cell bioavailability while maintaining Tsg101-binding affinity. We took note of the fact that cyclic versions of linear peptides can result in better cell-permeability. Additionally, appropriately restricting solution conformations through cyclization can also afford higher affinity.13–17 The objective of the current study was to apply RCM strategies to the WT 9-mer peptide and to observe the effects that such macrocyclization would have on Tsg101-binding affinity and cellular bioavailability.

Expression of the human epidermal growth factor receptor 2 (HER2) is amplified in 25 – 30% of breast cancers and has been associated with an unfavorable prognosis. Here we report the construction, purification, and characterization of Affitoxin – a novel class of HER2-specific cytotoxic molecules combining HER2-specific Affibody molecule as a targeting moiety and PE38KDEL, which is a truncated version of Pseudomonas exotoxin A (PE), as a cell killing agent. It is highly soluble and does not require additional refolding, oxidation, or reduction steps during its purification. Using Surface Plasmon Resonance (SPR) technology and competitive binding assays, we have shown that Affitoxin binds specifically to HER2 with nanomolar affinity. We have also observed a high correlation between HER2 receptor expression and retention of Affitoxin bound to the cell surface. Affitoxin binding and internalization is followed by PE activity domain-mediated ADP-ribosylation of translation elongation factor 2 (eEF2) and, consequently, inhibition of protein synthesis as shown by protein expression analysis of HER2-positive cells treated with Affitoxin. Measured IC50 value for HER2-negative cells MDA-MB468 (65±2.63 pM) was more than 20 times higher than the value for low HER2 level-expressing MCF7 cells (2.56±0.1 pM), and almost three orders of magnitude higher for its HER2-overexpressing derivative MCF7/HER2 (62.7±5.9 fM). These studies suggest that Affitoxin is an attractive PE38-based candidate for treatment of HER2-positive tumors.

Blocking the interaction between phosphotyrosine (pTyr)-containing activated receptors and the Src homology 2 (SH2) domain of the growth factor receptor-bound protein 2 (Grb 2) is considered to be an effective and non-cytotoxic strategy to develop new anti-proliferate agents due to its potential to shut down the Ras activation pathway. In this study, a series of phosphotyrosine containing cyclic pentapeptides were designed and synthesized based upon the phage library derived cyclopeptide, G1TE. A comprehensive SAR study was also carried out to develop potent Grb2-SH2 domain antagonists based upon this novel template. With both the peptidomimetic optimization of the amino acid side-chains and the constraint of the backbone conformation guided by molecular modeling, we developed several potent antagonists with low micromolar range binding affinity, such as cyclic peptide 15 with an Kd = 0.359 μM, which is providing a novel template for the development of Grb2-SH2 domain antagonists as potential therapeutics for certain cancers.

Expression of the retroviral Gag protein leads to formation of virus-like particles in mammalian cells. In vitro and in vivo experiments show that nucleic acid is also required for particle assembly. However, several studies have demonstrated that chimeric proteins in which the nucleocapsid domain of Gag is replaced by a leucine zipper motif can also assemble efficiently in mammalian cells. We have now analyzed assembly by chimeric proteins in which nucleocapsid of human immunodeficiency virus type 1 (HIV-1) Gag is replaced by either a dimerizing or a trimerizing zipper. Both proteins assemble well in human 293T cells; the released particles lack detectable RNA. The proteins can coassemble into particles together with full-length, wild-type Gag. We purified these proteins from bacterial lysates. These recombinant “Gag-Zipper” proteins are oligomeric in solution and do not assemble unless cofactors are added; either nucleic acid or inositol phosphates (IPs) can promote particle assembly. When mixed with one equivalent of IPs (which do not support assembly of wild-type Gag), the “dimerizing” Gag-Zipper protein misassembles into very small particles, while the “trimerizing” protein assembles correctly. However, addition of both IPs and nucleic acid leads to correct assembly of all three proteins; the “dimerizing” Gag-Zipper protein also assembles correctly if inositol hexakisphosphate is supplemented with other polyanions. We suggest that correct assembly requires both oligomeric association at the C terminus of Gag and neutralization of positive charges near its N terminus.

Hydrophobic active pharmaceutical ingredients (APIs) are often difficult to deliver effectively because of formulation limitations. Nanosuspensions of such drugs may be used to increase bioavailability and offer a variety of delivery options including injection, inhalation, oral, and transdermal. Microfluidics reaction technology (MRT) was used successfully to produce submicrometer API suspensions via a continuous process that involves solvent/antisolvent crystallization. As proof of concept, nanosuspensions of norfloxacin (NFN), an antibacterial agent, were produced varying the key parameters of the technology. The nanosuspensions had narrow particle size distributions and median particle sizes in the range of 170-350 nm. The particle size depends on the supersaturation ratio and energy dissipation expressed as processing pressure. However, the particle size was found to be insensitive to the presence of the surfactant used. The crystalline structure of NFN was not affected by the mixing intensity but by the solvent/antisolvent system. This "bottom up" process for creating nanosuspensions was compared to a "top down" process, in which NFN nanosuspensions were created as a result of particle size reduction. It was found that the "bottom up" process was substantially more efficient and resulted in smaller particles than the "top down" process. MRT is based on an impinging jet reactor design with jet velocities and energy dissipation that is orders of magnitude higher than those of conventional impinging jet reactors. The technology provides precise control of the feed rates and the subsequent location and intensity of mixing of the reactants. It may be the best choice economically due to its process intensification character that minimizes energy requirements and the proven scalability of the reactor.

The research examines viewers' actual responses to four televised fund-raising drives by a public television station over a 2-year period. The 584 pledge breaks we studied contain 4,868 individual appeals that were decomposed into two underlying dimensions based on the empathy-helping hypothesis: the appeal beneficiary (self versus other) and emotional valence (positive versus negative). We find that the most effective fund-raising appeals communicate the benefits to others rather than to the self and evoke negative rather than positive emotions. Appeals that emphasize benefits to the self significantly reduce the number of calls to the station, particularly when they have a positive emotional valence.

The approval of major infrastructure and industrial developments is often dependent on the results of environmental and social impact assessments (EIA and SIA, respectively). Depending on the recommendations of ‘experts’ undertaking the assessments, projects are approved, rejected or modified to take account of unintended negative consequences. In practice, there are pressures on consultants to come up with favourable results, and unfavourable results are often ignored. These pressures may be subtle. For example, because consultants are usually employed by somebody with an interest in seeing projects go ahead, any consultant with a reputation for being too negative is unlikely to get work. The present paper explores issues faced by anthropologists in SIA, as well as the structure of interests within which SIA is carried out, and ways in which ethical dilemmas can be negotiated. It asks whether simply opting out of the process altogether is an ethical option preferable to critical engagement.

HIV-1 viral assembly requires a direct interaction between a Pro-Thr-Ala-Pro (“PTAP”) motif in the viral protein Gag-p6 and the cellular endosomal sorting factor Tsg101. In an effort to develop competitive inhibitors of this interaction, an SAR study was conducted based on the application of post solid-phase oxime formation involving the sequential insertion of aminooxy-containing residues within a nonamer parent peptide followed by reaction with libraries of aldehydes. Approximately 15–20-fold enhancement in binding affinity was achieved by this approach.

In this paper we address the often sterile and circular debates over relationships between poverty and deforestation. These debates revolve around questions of whether forest loss causes poverty or poverty contributes to forest encroachment, and questions of whether it is loss of access to forests or dependence on forest-based livelihoods that cause poverty. We suggest that a way beyond the impasse is to set such debates within the context of agrarian change. Livelihoods of those who live in or near forests depend considerably on a rapidly changing agriculture, yet agrarian contexts receive only background attention in popular, political and academic discourse over poverty and forests. Moreover, to the extent that agriculture is considered, little heed is paid to social, technical and economic change. We therefore address agriculture's changing relationships with the wider economy, otherwise referred to as the agrarian transition, and with the natural resource base on which it depends. The paper draws on the experience of Thailand to illustrate our key argument, and more specifically addresses the situation on the resource periphery through a look at the agriculture-forest interface.

The stereoselective synthesis of aminooxy-containing proline analogues bearing Fmoc/Boc or Fmoc/Mtt protection that renders them suitable for incorporation into peptides using Fmoc protocols is reported. Acid-catalyzed unmasking at the completion of peptide synthesis yields free aminooxy-functionalities for oxime formation through reaction with libraries of aldehydes. This allows post solid-phase diversification strategies that may facilitate structure–activity relationship studies.

Ring-closing metathesis (RCM) was employed to join carboxy-terminal alkenyl glycine side chains together with vinyl- and allyl-functionality appended to the beta-methylene of amino-terminal phosphotyrosyl (pTyr) mimetics. This required the synthesis of a variety of new pTyr mimetics, including a novel aza-containing analogue. Many of the resulting 15-member macrocyclic tetrapeptide mimetics exhibited low nanomolar Grb2 SH2 domain-binding affinities in spite of the fact that differing ring junction stereochemistries and geometries of the RCM-derived double bond were employed. The finding that significant latitude exists in the structural requirements for ring closure may facilitate the development of therapeutically relevant macrocyle-based Grb2 SH2 domain-binding antagonists. The synthetic approaches used in this study may also find application to peptide mimetics directed at other biological targets.

After a service failure and a poor recovery, what leads loyal customers to try to punish a firm even if there is no material gain for doing so? We propose and test a justice-based model that incorporates perceived betrayal as the means to understand customer retaliation and the “love becomes hate” effect. The results suggest that betrayal is a key motivational force that leads customers to restore fairness by all means possible, including retaliation. In contrast to the majority of findings in the service literature, we propose and find that relationship quality has unfavorable effects on a customer’s response to a service recovery. As a relationship gains in strength, a violation of the fairness norm was found to have a stronger effect on the sense of betrayal experienced by customers. The model was tested on a national sample of airline passengers who complained to a consumer agency after an unsuccessful recovery.

Individual protein binding sites on DNA can be measured in bits of information. This information is related to the free energy of binding by the second law of thermodynamics, but binding kinetics appear to be inaccessible from sequence information since the relative contributions of the on- and off-rates to the binding constant, and hence the free energy, are unknown. However, the on-rate could be independent of the sequence since a protein is likely to bind once it is near a site. To test this, we used surface plasmon resonance and electromobility shift assays to determine the kinetics for binding of the Fis protein to a range of naturally occurring binding sites. We observed that the logarithm of the off-rate is indeed proportional to the individual information of the binding sites, as predicted. However, the on-rate is also related to the information, but to a lesser degree. We suggest that the on-rate is mostly determined by DNA bending, which in turn is determined by the sequence information. Finally, we observed a break in the binding curve around zero bits of information. The break is expected from information theory because it represents the coding demarcation between specific and nonspecific binding.

A 4-aminopiperidine-4-carboxylic acid residue was placed in the pTyr+1 position of a Grb2 SH2 domain-binding peptide to form a general platform, which was then acylated with a variety of groups to yield a library of compounds designed to explore potential binding interactions, with protein features lying below the betaD strand. The highest affinities were obtained using phenylethyl carbamate and phenylbutyrylamide functionalities.

One of the key elements in modern reservoir exploration and management is describing reservoir fluid phase behavior and physical properties commonly referred to as pressure-volume-temperature, or PVT data. Typically, PVT data come from laboratory tests, empirical correlations, and Equation of State (EOS) models. It is common practice to describe the phase behavior or PVT data through EOS models tuned to laboratory measurements on reservoir fluid samples. After a sample is received at a laboratory, a portfolio of PVT laboratory tests are performed. The results are quality checked and the appropriate data are selected to tune an EOS model to obtain an accurate EOS description of the reservoir fluid. Each step in this process requires judgment and decisions from the corresponding domain experts to achieve physically sound PVT relations and calculate the required properties. Such EOS based modelling processes are time consuming, expensive and exposed to various risks due to multiple human interventions. In the work presented in this paper, a study was conducted to explore the feasibility of a defined step EOS based modelling workflow using a limited, but defined, laboratory data set as the basis for characterizing and tuning the EOS model. In the development of a standardized workflow, a variety of EOS characterization and tuning methods were established to accommodate the diverse and complex nature of reservoir fluids. The methods considered include a modified Pedersen's method, a gamma distribution based method, and two methods based on single carbon number (SCN) composition and aromaticity factors. Despite their differences, the methods follow the principal objectives to be operator independent, robust, thermodynamically consistent, and numerically simple. Apart from the fluid composition, the only PVT data required for the proposed workflow were the saturation pressure, densities and the volumetric data obtained from constant composition expansion (CCE) measurements. The CCE test is advantageous because it is non-destructive to the sample being tested and can be performed quickly and reliably either in the laboratory or at a well-site. An optimized EOS model utilizing appropriate fluid characterization and tuning method is then selected based on a pre-defined Key Performance Indicator (KPI) derived from the deviation of the model predictions with the experimental data. With this optimal model defined, all other PVT data, such as those from sample destructive differential liberation (DL) or constant volume depletion (CVD) tests, can be reliably predicted. The workflow was validated using extensive PVT data measured for a variety of reservoir fluids including black oils, volatile oils, and gas condensates. The results have shown that the proposed workflow can reliably model PVT phase behavior and properties for the majority of tested reservoir fluids. This article will thoroughly discuss the details of this workflow and the modelling results on four benchmark fluids of different types.

Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.

Replacing the Pro6 in the p6(Gag)-derived 9-mer "P-E-P-T-A-P-P-E-E" with N-substituted glycine (NSG) residues is problematic. However, incorporation of hydrazone amides ("peptoid hydrazones") can be readily achieved in library fashion. Furthermore, reduction of these hydrazones to N-substituted "peptoid hydrazides" affords a facile route to library diversification. This approach is demonstrated by application to Tsg101-binding compounds designed as potential HIV budding antagonists. [reaction: see text]

Copper (I) promoted [3+2] Huisgen cycloaddition of azides with terminal alkynes was used to prepare triazole-containing macrocycles based on the Grb2 SH2 domain-binding motif, ‘Pmp-Ac6c-Asn’, where Pmp and Ac6c stand for 4-phosphonomethylphenylalanine and 1-aminocyclohexanecarboxylic acid, respectively. When cycloaddition reactions were conducted at 1 mM substrate concentrations, cyclization of monomeric units occurred. At 2 mM substrate concentrations the predominant products were macrocyclic dimers. In Grb2 SH2 domain-binding assays the monomeric (S)-Pmp-containing macrocycle exhibited a Kd value of 0.23 μM, while the corresponding dimeric macrocycle was found to have greater than 50-fold higher affinity. The open-chain dimer was also found to have affinity equal to the dimeric macrocycle. This work represents the first application of ‘click chemistry’ to the synthesis of SH2 domain-binding inhibitors and indicates its potential utility.

Partial differential equations and differential geometry come together in the idea of a generalized immersion. This concept, defined by means of Grassmann bundles and contact forms, allows for ?immersions? with ?singularities.? Sophus Lie?s generalized solutions to partial differential equations are an important special case. The classical second fundamental form has a natural generalization in the context of generalized immersions. The rank of the form is then meaningful. A constant rank assumption on the generalized second fundamental form leads to a natural foliation of the generalized immersion, at least when the ambient space is a space of constant curvature. Questions about the total geodesy and regularity of the foliation are also addressed.

Mass transfer experiments were conducted to determine the transport and interaction parameters of selected molecules in hydrated bacterial cellulose (BC) membranes. The objective was to determine physiochemical characteristics and elucidate the mechanisms governing transport in relation to the membrane structure. Pore and sorption models developed previously for the analysis of transport in hydrogel membranes were relevant to the cellulose membrane system, including: (1) interfacial phenomena between the bulk fluid and the outer membrane surfaces and/or along a pore wall, (2) sorption into the membrane matrix itself with diffusion possibly affected by immobilization at specific interactive sites, (3) free and/or fixed site diffusion within the matrix and if appropriate, through the porous regions, whether as distinct pores, micro-channels or other non-homogeneous/discrete areas and (4) chemical reactions that could alter the nature of the diffusing species or the media itself. Since all these mechanisms may be active, our classification, and thus characterization, was based upon whichever mechanism dominates. The diffusion coefficients of various dextrans (44–260 kDa), measured using a horizontal flow diffusion chamber, were compared to their corresponding values in water to demonstrate hindered diffusion. The degree of hindrance, ψ, was the same for all four dextrans indicating that the parameter is not a function of pore or micro-channel size but rather due to the presence of membrane fibers. Three representative marker molecules (Vitamin B12, lysozyme and bovine serum albumin) were evaluated using the same apparatus combined with desorption experiments to measure permeation (P) and effective diffusion (Deff) coefficients. Partition coefficients, H, were subsequently calculated and verified experimentally. Estimates of Deff in the BC membranes were made through a weighted average using literature values for the diffusion of the three molecules in water and regenerated cellulose along with measured H and ψ values. The experimental results are in excellent agreement with these estimates indicating the presence of dual transport mechanisms, for solute transport through the continuous water phase and cellulose matrix, with some hindrance of molecular diffusion via fiber obstruction. With Vitamin B12 and lysozyme (the two smallest solutes), equilibrium interactions such as adsorption and solubility are also important. These results help clarify the potential utility of these novel bio-derived membranes in a variety of possible separations scenarios.