Willingness to Pay for Eco‐Certified Refurbished Products: The Effects of Environmental Attitudes and Knowledge

Refurbishing products, which are increasingly sold in business‐to‐consumer markets, is a key strategy to reduce waste. Nevertheless, research finds that consumers’ willingness to pay (WTP) for refurbished products is low. Strategies for a higher WTP are needed in order to grow consumer markets for refurbished products. Eco‐certification of refurbished products may be a key strategy here. Drawing on the consumer WTP literature concerning “green” products, we investigate the impact of independent eco‐certificates. Our analysis is based on a survey of 231 potential customers. The results suggest that, across various product categories, the WTP for products with refurbished components is significantly lower. Adding an eco‐certificate tends to return the WTP toward the virgin product level. We show that consumers with proenvironmental attitudes particularly exhibit green buying behavior. Our findings indicate that eco‐certification is often worthwhile because it enhances the business rationale for producing products with refurbished components.     

Bioanalytical strategy used in development of pharmacokinetic (PK) methods that support biosimilar programs

The development of biosimilar products is expected to grow rapidly over the next five years as a large number of approved biologics reach patent expiry. The pathway to regulatory approval requires that similarity of the biosimilar to the reference product be demonstrated through physiochemical and structural characterization, as well as within in vivo studies that compare the safety and efficacy profiles of the products. To support nonclinical and clinical studies pharmacokinetic (PK) assays are required to measure the biosimilar and reference products with comparable precision and accuracy. The most optimal approach is to develop a single PK assay, using a single analytical standard, for quantitative measurement of the biosimilar and reference products in serum matrix. Use of a single PK assay for quantification of multiple products requires a scientifically sound testing strategy to evaluate bioanalytical comparability of the test products within the method, and provide a solid data package to support the conclusions. To meet these objectives, a comprehensive approach with scientific rigor was applied to the development and characterization of PK assays that are used in support of biosimilar programs. Herein we describe the bioanalytical strategy and testing paradigm that has been used across several programs to determine bioanalytical comparability of the biosimilar and reference products. Data from one program is presented, with statistical results demonstrating the biosimilar and reference products were bioanalytically equivalent within the method. The cumulative work has established a framework for future biosimilar PK assay development.     

Bioanalytical strategy used in development of pharmacokinetic (PK) methods that support biosimilar programs

The development of biosimilar products is expected to grow rapidly over the next five years as a large number of approved biologics reach patent expiry. The pathway to regulatory approval requires that similarity of the biosimilar to the reference product be demonstrated through physiochemical and structural characterization, as well as within in vivo studies that compare the safety and efficacy profiles of the products. To support nonclinical and clinical studies pharmacokinetic (PK) assays are required to measure the biosimilar and reference products with comparable precision and accuracy. The most optimal approach is to develop a single PK assay, using a single analytical standard, for quantitative measurement of the biosimilar and reference products in serum matrix. Use of a single PK assay for quantification of multiple products requires a scientifically sound testing strategy to evaluate bioanalytical comparability of the test products within the method, and provide a solid data package to support the conclusions. To meet these objectives, a comprehensive approach with scientific rigor was applied to the development and characterization of PK assays that are used in support of biosimilar programs. Herein we describe the bioanalytical strategy and testing paradigm that has been used across several programs to determine bioanalytical comparability of the biosimilar and reference products. Data from one program is presented, with statistical results demonstrating the biosimilar and reference products were bioanalytically equivalent within the method. The cumulative work has established a framework for future biosimilar PK assay development.     

The use of hydrocarbon analyses for environmental assessment and remediation

Battelle Ocean Sciences has developed an analytical approach to identify and’ quantify petroleum products, coal products, and individual hydrocarbon components at trace levels in complex environmental matrices. The hydrocarbon analysis strategy uses capillary gas chromatography/flame ionization detection for alkane and total oil analysis, combined with gas chromatography/mass spectrometry for polynuclear aromatic hydrocarbon analysis. The method provides environmentally realistic analyte detection limits (parts per trillion in water, parts per billion in sediments) and an analyte list that is designed specifically for petroleum and coal‐based products. Results are compared to a detailed computerized library of total, water‐soluble, and degraded hydrocarbon products. The systematic data interpretation strategy maximizes the accuracy of petroleum and coal product identification in environmental matrices and represents a vast improvement over standard EPA methodology.     

Application of pyramided traits against Lepidoptera in insect resistance management for Bt crops

Since initial launch of insect protected transgenic crops, the most effective strategy to manage the potential for target pests to evolve resistance has been the use of a single mode of action with "high dose" and structured refuge. However, the effectiveness of this strategy is limited if mortality of certain pests does not reach "high dose" criteria, inconsistent implementation of refuges and non-rare resistance alleles. More recently, several pyramided trait products, which include multiple modes of action against key target pests, have been developed. These products offer the potential for dramatically improved resistance management with smaller refuges and less dependence on high mortality of susceptible and heterozygous insects and rare resistance alleles. We show that products such as SmartStax and PowerCore offer compelling resistance management benefits compared with single mode of action products and allow for the option of products containing refuge seed mixtures rather than structured refuges to effectively delay resistance. We conclude that all stakeholders, including technology developers, growers, crop advisors, extensions services and regulatory authorities should continue to encourage the development, deployment and adoption of pyramided trait products for improved pest management and improved resistance management.     

天然产物的糖基化及糖基侧链改造

抗生素和抗癌药物等多种天然产物的活性都依赖于其糖基侧链,糖基侧链结构的变化对母体化合物的生物活性、底物适应性及药理学性质具有重要影响。糖基侧链结构变化多端,修饰、改变天然产物的糖基侧链已成为获得临床候选药物的重要方法。利用化学法和酶法,研究者创造了多种改造天然产物糖基化的方法。详细介绍了天然产物的糖基化过程,并从组合生物学、糖基转移酶改造、糖类随机化及新型糖类随机化和糖基转移酶可逆性四方面阐述了糖基侧链的改造方法。     

A cDNA library functional screening strategy based on fluorescent protein complementation assays to identify novel components of signaling pathways

Progress towards a deeper understanding of cellular biochemical networks demands the development of methods to both identify and validate component proteins of these networks. Here, we describe a cDNA library screening strategy that achieves these aims, based on a protein-fragment complementation assay (PCA) using green fluorescent protein (GFP) as a reporter. The strategy combines a simple cell-based cDNA-screening approach (interactions of a "bait" protein of interest with "prey" cDNA products) with specific functional assays that use the same system and provide initial validation of the cDNA products as being biologically relevant. We applied this strategy to identify novel interacting partners of the protein kinase PKB/Akt. This method provides very general means of identifying and validating genes involved in any cellular process and is particularly designed for identifying enzyme substrates or regulatory proteins for which the enzyme specificity can only be defined by their interactions with other proteins in cells in which the proteins are normally expressed.     

Implications of Livelihood Dependence on Non-Timber Products in Peruvian Amazonia

The present and future well-being of the world’s forest dwelling populations depends on their ability to gain livelihood resources from their immediate environment. Sustainable extraction of non-timber forest products has been promoted by conservationists and development agencies as a feasible strategy for forest dwellers that does not compromise the resource base. Yet surveys of actual resource use suggest that for poorer resource-dependent communities without access to markets, non-timber forest products can only ever represent a safety-net activity and a supplementary income source. Others argue that resource availability, in terms of the diversity and productivity of the forest, is the key parameter in realizing a contribution of forest products to well-being. This paper examines the scope and heterogeneity of forest product use to reveal whether resource availability necessarily provides the context for significant contributions to well-being of forest dwellers. We present data from an area of tropical rainforest, close to Iquitos in Peru, which was previously shown to have high potential value. We find, through a census survey of households within a forest reserve area, that non-timber forest products provide only a relatively small portion of income and that only a small proportion of available products are actually commercialized, despite apparent market availability. We show that the low rates of commercialization can be explained by unequal access capital assets used for extraction, to natural resources themselves, and to product markets. They are also explained by the concentration of capital-poor households on subsistence gathering activities. The value of destructive uses of forests, both logging and agriculture, remain higher than returns from non-timber products. This research demonstrates that although non-timber forest products are an important livelihood source, market integration and commercialization is not everywhere an appropriate or realistic strategy.     

A series of novel directional cloning and expression vectors for blunt-end ligation of PCR products

Novel directional cloning and expression vectors were developed for blunt-end ligation of PCR products that are suitable for high-throughput cloning and simplifying the screening procedure. The PCR products, without further processing, are cloned into vectors digested with SchI and, following transformation, the desired recombinants give typical blue colonies on selectable plates. The principle of this selection strategy is that the construction also generates a full-length ideal lacO gene. To the best of our knowledge, this is the first time that this lacO reconstruction strategy has been applied in the selection of recombinants.     

CrossSearch, a user-friendly search engine for detecting chemically cross-linked peptides in conjugated proteins

Chemical cross-linking and high resolution MS have been integrated successfully to capture protein interactions and provide low resolution structural data for proteins that are refractive to analyses by NMR or crystallography. Despite the versatility of these combined techniques, the array of products that is generated from the cross-linking and proteolytic digestion of proteins is immense and generally requires the use of labeling strategies and/or data base search algorithms to distinguish actual cross-linked peptides from the many side products of cross-linking. Most strategies reported to date have focused on the analysis of small cross-linked protein complexes (<60 kDa) because the number of potential forms of covalently modified peptides increases dramatically with the number of peptides generated from the digestion of such complexes. We report herein the development of a user-friendly search engine, CrossSearch, that provides the foundation for an overarching strategy to detect cross-linked peptides from the digests of large (>or=170-kDa) cross-linked proteins, i.e. conjugates. Our strategy combines the use of a low excess of cross-linker, data base searching, and Fourier transform ion cyclotron resonance MS to experimentally minimize and theoretically cull the side products of cross-linking. Using this strategy, the (alpha beta gamma delta)(4) phosphorylase kinase model complex was cross-linked to form with high specificity a 170-kDa betagamma conjugate in which we identified residues involved in the intramolecular cross-linking of the 125-kDa beta subunit between its regulatory N terminus and its C terminus. This finding provides an explanation for previously published homodimeric two-hybrid interactions of the beta subunit and suggests a dynamic structural role for the regulatory N terminus of that subunit. The results offer proof of concept for the CrossSearch strategy for analyzing conjugates and are the first to reveal a tertiary structural element of either homologous alpha or beta regulatory subunit of phosphorylase kinase.     

Protein structure similarity clustering (PSSC) and natural product structure as inspiration sources for drug development and chemical genomics

Finding small molecules that modulate protein function is of primary importance in drug development and in the emerging field of chemical genomics. To facilitate the identification of such molecules, we developed a novel strategy making use of structural conservatism found in protein domain architecture and natural product inspired compound library design. Domains and proteins identified as being structurally similar in their ligand-sensing cores are grouped in a protein structure similarity cluster (PSSC). Natural products can be considered as evolutionary pre-validated ligands for multiple proteins and therefore natural products that are known to interact with one of the PSSC member proteins are selected as guiding structures for compound library synthesis. Application of this novel strategy for compound library design provided enhanced hit rates in small compound libraries for structurally similar proteins.     

Development of a plasmepsin II fluorescence polarization assay suitable for high throughput antimalarial drug discovery

Despite decades of research, malaria remains the world's most deadly parasitic disease. New treatments with novel mechanisms of action are urgently needed. Plasmepsin II is an aspartyl protease that has been validated as an antimalarial therapeutic target enzyme. Although natural products form the basis of most modern antimalarial drugs, no systematic high-throughput screening has been reported against this target. We have designed an effective strategy for carrying out high-throughput screening of an extensive library of natural products that uses a fluorescence resonance energy transfer primary screening assay in tandem with a fluorescence polarization assay. This strategy allows rapid screening of the library coupled with effective discrimination and elimination of false-positive samples and selection of true hits for chemical isolation of inhibitors of plasmepsin II.     

PCR-free mutation detection of BRCA1 on a zip-code microarray using ligase chain reaction

We describe here ligation-based strategy to detect mutations in BRCA1 utilizing zip-code microarray technology. In our first approach, PCR was performed to amplify the genomic regions containing the mutation sites. The PCR products were then used as templates in a subsequent ligation reaction using two ligation primers that flanked the mutation site. The primary allele-specific primer is designed to contain a base of mutation site at its 3′ end with 5′ complementarity to the respective zip-code sequence while the secondary common primer is modified by biotin at its 3′ end. Depending on the genotype of samples at the mutation site, the nick between the two ligation primers can be sealed in the presence of DNA ligase. The ligation products were then hybridized on the zip-code microarray followed by staining with streptavidine-cy3 to generate a fluorescent signal. Using this strategy we successfully genotyped selected Korean-specific mutation sites in exon 11 of BRCA1 with a wild type and two heterozygote mutant samples. Furthermore, we also demonstrated that ligase chain reaction using unamplified genomic DNA as direct templates is enough to generate sufficient signals for correct genotypings in a multiplexed manner, verifying first that PCR is not essential for this microarray-based strategy.     

The activity of human CYP2D6 in low water organic solvents

P450 enzymes are of high interest for synthetic applications due to their ability to catalyze hydroxylation reactions at inactivated C-H bonds. The low solubility of many substrates in buffer, however, is limiting the applications of P450s. Our recent demonstration that the P450 enzymes CYP2D6 and CYP3A4 can function very well in biphasic solvent systems is one step towards overcoming this drawback, but is not practical when substrates or products are unstable in water, or with water-soluble products. An alternative strategy, which also facilitates enzyme recycling, is to directly resuspend lyophilized enzyme into nearly anhydrous organic solvents. Interestingly, we report here that CYP2D6 colyophilized with trehalose and suspended in n-decane shows higher activity than in aqueous buffer. This study demonstrates the unexpected high tolerance of CYP2D6 to some low water organic solvents and provides an alternative strategy to facilitate the use of this enzyme in synthesis.     

Quick and clean cloning: a ligation-independent cloning strategy for selective cloning of specific PCR products from non-specific mixes

We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. The reaction mix is then directly transformed into E. coli where the annealed vector-insert complex is repaired and ligated. We have tested this method, which we call quick and clean cloning (QC cloning), for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity.     

Efficient production of oxidized terpenoids via engineering fusion proteins of terpene synthase and cytochrome P450

The functionalization of terpenes using cytochrome P450 enzymes is a versatile route to the production of useful derivatives that can be further converted to value-added products. Many terpenes are hydrophobic and volatile making their availability as a substrate for P450 enzymes significantly limited during microbial production. In this study, we developed a strategy to improve the accessibility of terpene molecules for the P450 reaction by linking terpene synthase and P450 together. As a model system, fusion proteins of 1,8-cineole synthase (CS) and P450_cin were investigated and it showed an improved hydroxylation of the monoterpenoid 1,8-cineole up to 5.4-fold. Structural analysis of the CS-P450_cin fusion proteins by SEC-SAXS indicated a dimer formation with preferred orientations of the active sites of the two domains. We also applied the enzyme fusion strategy to the oxidation of a sesquiterpene epi-isozizaene and the fusion enzymes significantly improved albaflavenol production in engineered E. coli. From the analysis of positive and negative examples of the fusion strategy, we proposed key factors in structure-based prediction and evaluation of fusion enzymes. Developing fusion enzymes for terpene synthase and P450 presents an efficient strategy toward oxidation of hydrophobic terpene compounds. This strategy could be widely applicable to improve the biosynthetic titer of the functionalized products from hydrophobic terpene intermediates.     

A transposon-based strategy to scale up myxothiazol production in myxobacterial cell factories

Myxobacteria are proficient producers of biologically active secondary metabolites. However, efforts to exploit these natural products for the development of new therapeutics and agrochemicals are frequently hampered by low production levels. We describe here a transposon-based strategy to identify genes encoding regulators of secondary metabolite biosynthesis in the myxobacterium Angiococcus disciformis An d48, which produces the highly efficient electron transport inhibitor myxothiazol. Extracts from 1200 transposon mutants were analyzed by HPLC, leading to the identification of six mutants in which myxothiazol production was increased by as much as 30-fold. Identifying the sites of integration coupled with sequencing of flanking regions, showed that some of the inactivated genes encode proteins with similarity to known bacterial regulators such as two-component systems and serine-threonine protein kinases. However, other gene products do not resemble any characterized proteins. Taken together, these data show that this transposon-based strategy is a valuable tool to identify regulatory genes of secondary metabolism, including gene loci which cannot be detected using current in silico approaches.     

EFFECT OF ASSESSORS EXPERTISE LEVEL ON EFFICIENCY OF WARM-UP FOR TRIANGLE TESTS

The effect of warm-up on performance for repeated triangle tests is studied according to assessors' expertise level for both triangle test strategy and the pair of products to compare. Three experiments performed with orange flavored soft drinks show that the effect of warm-up depends on the assessors' expertise: (1) naive assessors do not increase their performance with warm-up; (2) assessors with a moderate practice of both triangle tests and the pair of products improve their performance with warm-up; (3) assessors with a moderate practice of triangle tests, but not familiar with the pair of products, improve their performance with warm-up too; and (4) assessors highly experienced for both triangle tests and products do not improve their performance with warm-up. These results support the idea that the positive effect of warm-up is due to an attentional process: Warm-up seems to help assessors focusing their attention on the dimension on which the two products are actually different and ignoring the other dimensions. Thus, results show that assessors are able to learn the difference between the two products and to learn to focus their attention on this difference at any time. They also suggest that prior knowledge of the triangle test scheme is required to benefit from warm-up.     

Mild Detritylation of Nucleic Acid Hydroxyl Groups by Warming Up

It is challenging to effectively deprotect hydroxyl groups of acid-or-base sensitive bio-macromolecules without causing even minor defects and compromising high quality of final products. We report here a mild detritylation strategy in mildly acidic buffers to remove the DMTr protection from the 5′-hydroxyl groups of synthetic nucleic acids. The DMTr-groups can be easily and effectively removed at pH 4.5 or 5.0 with slight warming up (40°C), offering virtually quantitative deprotection. This warming-up strategy is particularly useful for deprotection of the modified nucleic acids that are sensitive to the conventional acid deprotection. As a first step towards our long-term goal of synthesizing defect-free nucleic acids, our novel and simple strategy further increases the quality of synthetic nucleic acids.