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Abstract In two studies, we introduce the concept of spontaneous side-taking (SST) to describe how people initially align themselves in a conflict. The effects of side-taking in established conflicts are well studied, such as empathetic engagement and polarization. However, there is less known about how people spontaneously choose sides in situations without prior allegiances. To study these neglected effects of SST, we use two-character narratives to present participants with new conflicts in which they have no initial vested interest. These short narratives describe acts of aggression and detail the victimization of one character by another. Through two studies, we find: (1) victim characters receive greater attention and are remembered better; (2) victim characters receive more empathy; (3) victim characters are more likely to be viewed as the authors of conflict narratives; (4) and that side-taking flexibility diminishes after the first side-taking choice. This last finding means that there is a tendency for SST to stick over time. Overall, these findings demonstrate how victimization triggers and sustains side-taking in narratives by enhancing memory of and empathy for the victim.

Abstract We compare how humans retell stories to how ChatGPT retells stories in chains of three retellings by different people or different accounts on ChatGPT. ChatGPT provides competent summaries of the original narrative texts in one step of retelling. In subsequent retellings few additional changes occur. Human retellers, by contrast, reduce the original text incrementally and by creating 55–60% of novel words and concepts (synsets) at each iteration. The retellings by both ChatGPT and humans show very stable emotion ratings, which is a puzzle for human retellers given the high degree of novel inventions across retellings. ChatGPT maintains more nouns, adjectives, and prepositions and also uses language later acquired in life, while humans use more verbs, adverbs, and negations and use language acquired at a younger age. The results reveal that spontaneous retelling by humans involves ongoing creativity, anchored by emotions, beyond the default probabilistic wording of large language models such as ChatGPT.

Illicit financial flows (IFFs) deprive low-income countries of essential revenues while donors’ willingness to fund aid budgets dwindles. IFFs related to foreign direct investment and trade include transfer mispricing, trade mispricing and profit shifting. Policy options to curb IFFs range from short-term fixes to mid-term measures that adjust legal instruments and improve coordination between countries, to more fundamental structural reforms that require a longer time horizon. Which policies are effective and should be pursued is a highly contested point, slowing down the progress of reform. This is unsurprising as reducing IFFs involves a distributional conflict: more for those deprived of revenues now means less for those who currently benefit. We conduct a Q-methodology study among IFF policy experts. We use Q-methodology to reveal participants’ policy preferences and tease out lines of contestation and areas of agreement to identify the policy space available in which to advance reform. We find tensions existing amid preferences for short-term fixes and for more comprehensive structural reforms; tensions regarding the question of extending legal liability to those facilitating and assisting in the creation of IFFs; and tensions over whether and to what extent host countries should be empowered to curb IFFs using their legislative sovereignty. Policy measures to increase targeted transparency that is directly actionable to tax administrations in host countries are the most likely to garner approval from all stakeholders.

Establishing a more transparent, effective and equitable and framework for trade and taxation is crucial to enabling commodity exporting states to mobilise domestic resources for sustainable development. The current drive to reform the global governance of taxation offers opportunities, but takes place in the context of heightened North–South tensions, reduced trust in multilateralism and calls for deeper decolonisation. Drawing on a six-year multidisciplinary research project involving academic institutions from commodity exporting and trading countries, this chapter presents a research framework that the authors used for the study of illicit financial flows associated with commodity trade. It discusses major findings, and recommendations as to how to counter the ensuing tax base erosion in resource-rich developing countries. The latter can consider a range of policies and innovative measures to rein in commodity trade mispricing. But this is not enough. States hosting major trading and financial centres have to simultaneously address a range of pull factors. At the global level, fair taxation reform is key and must preserve a sovereign policy space, in which commodity exporting states may adopt context-specific solutions aligned with their institutional capacities. Finally, this chapter introduces the thematic volume of International Development Policy on illicit financial flows in the commodity sector and beyond.

Abstract Story retelling is a fundamental medium for the transmission of information between individuals and among social groups. Besides conveying factual information, stories also contain affective information. Though natural language processing techniques have advanced considerably in recent years, the extent to which machines can be trained to identify and track emotions across retellings is unknown. This study leverages the powerful RoBERTa model, based on a transformer architecture, to derive emotion-rich story embeddings from a unique dataset of 25,728 story retellings. The initial stories were centered around five emotional events (joy, sadness, embarrassment, risk, and disgust—though the stories did not contain these emotion words) and three intensities (high, medium, and low). Our results indicate (1) that RoBERTa can identify emotions in stories it was not trained on, (2) that the five emotions and their intensities are preserved when they are transmitted in the form of retellings, (3) that the emotions in stories are increasingly well-preserved as they experience additional retellings, and (4) that among the five emotions, risk and disgust are least well-preserved, compared with joy, sadness, and embarrassment. This work is a first step toward quantifying situation-driven emotions with machines.

Abstract Background Addressing the uptake of research findings into policy-making is increasingly important for researchers who ultimately seek to contribute to improved health outcomes. The aims of the Swiss Programme for Research on Global Issues for Development (r4d Programme) initiated by the Swiss National Science Foundation and the Swiss Agency for Development and Cooperation are to create and disseminate knowledge that supports policy changes in the context of the 2030 Agenda for Sustainable Development. This paper reports on five r4d research projects and shows how researchers engage with various stakeholders, including policy-makers, in order to assure uptake of the research results. Methods Eleven in-depth interviews were conducted with principal investigators and their research partners from five r4d projects, using a semi-structured interview guide. The interviews explored the process of how stakeholders and policy-makers were engaged in the research project. Results Three key strategies were identified as fostering research uptake into policies and practices: (S1) stakeholders directly engaged with and sought evidence from researchers; (S2) stakeholders were involved in the design and throughout the implementation of the research project; and (S3) stakeholders engaged in participatory and transdisciplinary research approaches to coproduce knowledge and inform policy. In the first strategy, research evidence was directly taken up by international stakeholders as they were actively seeking new evidence on a very specific topic to up-date international guidelines. In the second strategy, examples from two r4d projects show that collaboration with stakeholders from early on in the projects increased the likelihood of translating research into policy, but that the latter was more effective in a supportive and stable policy environment. The third strategy adopted by two other r4d projects demonstrates the benefits of promoting colearning as a way to address potential power dynamics and working effectively across the local policy landscape through robust research partnerships. Conclusions This paper provides insights into the different strategies that facilitate collaboration and communication between stakeholders, including policy-makers, and researchers. However, it remains necessary to increase our understanding of the interests and motivations of the different actors involved in the process of influencing policy, identify clear policy-influencing objectives and provide more institutional support to engage in this complex and time-intensive process.

Industrial mining transforms local landscapes, including important health determinants like clean water and sanitation. In this paper, we combined macro-level quantitative and micro-level qualitative data to show how mining projects affect water infrastructures and ultimately the health of affected communities. Although we observed a positive trend of water infrastructure in mining settings, surrounding communities are also characterized by water scarcity and degradation of water quality. The video at the core of this publication showcases inter-linkages of the findings obtained at both the macro- and the micro-levels, embedding our results in a geospatial context. While mining projects can have positive impacts on the development of local water infrastructure, improved management of negative impacts of mining projects is needed for promoting ‘Good health and well-being’ and ‘Clean water and sanitation’ as promulgated by the Sustainable Development Goals of the 2030 Agenda.

Droplet size, which is affected by nozzle type, nozzle setups and operation, and spray solution, is one of the most critical factors influencing spray performance, environment pollution, food safety, and must be considered as part of any application scenario. Characterizing spray nozzles can be a timely and expensive proposition if the entire operational space (all combinations of spray pressure and orifice size, what influence flow rate) is to be evaluated. This research proposes a structured, experimental design that allows for the development of computational models for droplet size based on any combination of a nozzle’s potential operational settings. The developed droplet size determination model can be used as Decision Support System (DSS) for precise selection of sprayer working parameters to adapt to local field scenarios. Five nozzle types (designs) were evaluated across their complete range of orifice size (flow rate*) and spray pressures using a response surface experimental design. Several of the models showed high level fits of the modeled to the measured data while several did not as a result of the lack of significant effect from either orifice size (flow rate*) or spray pressure. The computational models were integrated into a spreadsheet based user interface for ease of use. The proposed experimental design provides for efficient nozzle evaluations and development of computational models that allow for the determination of droplet size spectrum and spraying classification for any combination of a given nozzle’s operating settings. The proposed DSS will allow for the ready assessment and modification of a sprayers performance based on the operational settings, to ensure the application is made following recommendations in plant protection products (PPP) labels.

The UK’s Defence Science and Technology Laboratory (Dstl) is partnering with the US Naval Research Laboratory (NRL) on a joint mission to launch miniature sensors that will advance space weather measurement and modelling capabilities. The Coordinated Ionospheric Reconstruction Cubesat Experiment (CIRCE) comprises two 6U cube-satellites that will be launched into a near-polar low earth orbit (LEO), targeting 500 km altitude, in 2021. The UK contribution to CIRCE is the In situ and Remote Ionospheric Sensing (IRIS) suite, complementary to NRL sensors, and comprising three highly miniaturised payloads provided to Dstl by University College London (UCL), University of Bath, and University of Surrey/Surrey Satellite Technology Ltd (SSTL). One IRIS suite will be flown on each satellite, and incorporates an ion/neutral mass spectrometer, a tri-band global positioning system (GPS) receiver for ionospheric remote sensing, and a radiation environment monitor. From the US, NRL have provided two 1U Triple Tiny Ionospheric Photometers (Tri-TIPs) on each satellite (Nicholas et al., 2019), observing the ultraviolet 135.6 nm emission of atomic oxygen at night-time to characterize the two-dimensional distribution of electrons.

We use an interactive story design in which participants read short stories and make two consecutive plot choices about whether protagonists commit low- or high-violence actions. Our study has four main findings. 1) People who choose high violence report greater satisfaction with the story, while those switching to or staying with no violence show lower satisfaction. 2) However, when participants encounter these stories without choices, they reliably rate higher-violence stories as less satisfying than lower-violence stories. 3) Regret seems to account for the low satisfaction of those who choose or switch to low violence. 4) There is a large segment of people (up to 66%) who can be persuaded by different story contexts (genre, perspective) to choose extreme violence in interactive fiction and as a consequence of their choice feel satisfaction. We hypothesize that people who opt for high violence enjoy the story as a result of their choice. Overall, we suggest that choosing violence serves as a gateway for enjoyment by creating an aesthetic zone of control detached from morality.

When people retell stories, what guides their retelling? Most previous research on story retelling and story comprehension has focused on information accuracy as the key measure of stability in transmission. This paper suggests that there is a second, affective, dimension that provides stability for retellings, namely the audience affect of surprise. In a large-sample study with multiple iterations of retellings, we found evidence that people are quite accurate in preserving all degrees of surprisingness in serial reproduction – even when the event that produced the surprisingness in the original story is dropped or changed. Thus, we propose that the preservation of affect is an implicit goal of retelling: merely do retellers not recall highly surprising events better, but rather they register all levels of surprisingness precisely and aim to surprise their implied audience to same degree. This study used 2,389 participants.Significance Statement: Story retelling is a process whereby cultural information is transmitted horizontally across social networks and vertically down generations. For the most part, retelling research has focused on the relevance and stability of factual information, “who did what, where, when, and why”; comparatively little is known about the transmission of affective information. We suggest that affect can serve as a second axis of stability for retelling, partially independent from factual information. In serial reproduction tasks modeled after the telephone game, we find that surprisingness of stories is well preserved across retellings – even when the facts and events of the story are not. The findings are significant for the communication of information, and thereby also the stability and transformation of culture in general.

In-field measures of physical spray concentration do not tend to correlate well with caged insect mortality data. This is partly due to the reduced penetration of the spray into the cage. Spray penetration is hindered by the structure of the cage. Wind tunnel studies were conducted to investigate the accuracy of those calculations developed to correct for filtration levels in caged mosquito bioassays. Zenivex E20 (Etofenprox) was applied at rates ranging from an LD10 to an LD90. Three cage types were used, each with different penetration levels. The dose approaching the cage was converted to the dose entering the cage using cage penetration data from previous research. The penetration conversion factor returned a data set that directly correlated dose with mosquito mortality (R2 = = 0.918). The mortality percent was a function of the dose within the cage. The mesh type acted as a regulator. Although the conversion factor was effective, the differences between cages was not always significant due to within-group variation.

Do windfalls from the extractive sector help developing countries invest in human capital? To date, empirical studies remain inconclusive. Using Bolivia as a case study, this chapter examines the specific political-economic dynamics that led the country to increase spending on education yet at the same time failed to build a skilled workforce. Overall, the study finds that while the mining sector continued to seek unskilled, cheap labour, the capital-intensive hydrocarbon sector, for its part, developed on-the-job training programmes. Meanwhile, education policies failed to anticipate evolving demand from the labour market. As a result, vocational training suffered, a situation further compounded by efforts of powerful groups in the education sector to protect their own, somewhat narrow interests, at the expense of educational achievements. It concludes that the rise of private education and popular skills-based training programmes cannot substitute for development of a functional vocational training system, capable of supporting the country’s ambition to develop a world-class lithium processing industry with linkages in strategic sectors.

Sigurd Allern (2001) Nyhetsverdier. Om markedsorientering og journalistikk i ti norske aviser. Kristiansand: IJ-forlaget.

Developing an effective mRNA therapeutic often requires maximizing protein output per delivered mRNA molecule. We previously found that coding sequence (CDS) design can substantially affect protein output, with mRNA variants containing more optimal codons and higher secondary structure yielding the highest protein outputs due to their slow rates of mRNA decay. Here, we demonstrate that CDS-dependent differences in translation initiation and elongation rates lead to differences in translation- and deadenylation-dependent mRNA decay rates, thus explaining the effect of CDS on mRNA half-life. Surprisingly, the most stable and highest-expressing mRNAs in our test set have modest initiation/elongation rates and ribosome loads, leading to minimal translation-dependent mRNA decay. These findings are of potential interest for optimization of protein output from therapeutic mRNAs, which may be achieved by attenuating rather than maximizing ribosome load., Competing Interests: Declaration of interests D.W.R., A.A.B., M.C.L., A.K.J., Y.M.C., M.L., C.J.H., R.J., E.A.A., M.M., and B.R.F. are employees of Moderna, Inc., and hold stock/stock options in the company. C.K. and C.S.P. are former employees of Moderna, Inc., and hold stock/stock options in the company. M.J.M. is a former employee of Moderna, Inc., and a current consultant, and holds stock/stock options in the company., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: This study sought to determine whether firocoxib (FIRO) or meloxicam (MEL) was effective at providing analgesia after surgical castration in goats., Animals: 18 intact male crossbred goats (6 to 8 months old) were enrolled with a mean weight of 32.6 (± 2.9) kg., Methods: Surgical castration was done under injectable anesthesia by a licensed veterinarian. Twelve bucks were surgically castrated and given either FIRO (n = 6) or MEL (n = 6). Six bucks served as controls (CNTLs) and were not castrated. Outcome measurements included visual analogue scale, infrared thermography, plasma cortisol, plasma substance P, and kinetic gait analysis. All outcome measurements were obtained at -24, 4, 8, 24, 48, and 72 hours., Results: All 3 treatments were significantly different from each other at the 24- and 48-hour time points, with MEL animals having lower visual analogue scale scores when compared to FIRO animals; CNTL animals exhibited the lowest plasma cortisol levels (3.19 ng/mL; 95% CI, -1.21 to 7.59 ng/mL) followed by FIRO (7.45 ng/mL; 95% CI, 3.10 to 11.80 ng/mL) and MEL (10.24 ng/mL; 95% CI, 5.87 to 14.60 ng/mL). FIRO had an average mean decrease in gait velocity change (-54.17 cm/s; 95% CI, -92.99 to -15.35 cm/s), while MEL had an increase in gait velocity when compared to baseline values (14.54 cm/s; 95% CI, -24.27 to 53.36 cm/s). Control animals had an average mean of -3.06 cm/s (95% CI, -41.88 to 35.75 cm/s)., Clinical Relevance: Results from this study showed that there were some analgesic effects from administering MEL when compared to bucks that received a placebo treatment (CNTL).

Colonization of the gut and airways by pathogenic bacteria can lead to local tissue destruction and life-threatening systemic infections, especially in immunologically compromised individuals. Here, we describe an mRNA-based platform enabling delivery of pathogen-specific immunoglobulin A (IgA) monoclonal antibodies into mucosal secretions. The platform consists of synthetic mRNA encoding IgA heavy, light, and joining (J) chains, packaged in lipid nanoparticles (LNPs) that express glycosylated, dimeric IgA with functional activity in vitro and in vivo. Importantly, mRNA-derived IgA had a significantly greater serum half-life and a more native glycosylation profile in mice than did a recombinantly produced IgA. Expression of an mRNA encoded Salmonella-specific IgA in mice resulted in intestinal localization and limited Peyer's patch invasion. The same mRNA-LNP technology was used to express a Pseudomonas-specific IgA that protected from a lung challenge. Leveraging the mRNA antibody technology as a means to intercept bacterial pathogens at mucosal surfaces opens up avenues for prophylactic and therapeutic interventions., Competing Interests: Declaration of interests C.E.D., A.F.R., T.Y., M.J.M., Z.W., Y.-T.L., B.R.F., S.H., D.L., R.K., S.L., C.J.H., I.L.R., H.K., M.K., A.C., and O.J.P. are employees of and shareholders in Moderna, Inc. C.E.D. and O.J.P. are co-inventors on international patent WO 2022/212191 A1. E.N. was an employee of and shareholder in Moderna Inc. at the time of the study., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lameness continues to be a critical health and welfare concern associated with goat production. Amphotericin B (amp B) is an antimicrobial successful in inducing transient lameness for research purposes previously in livestock animals. The objectives of this study were to (1) identify which of three varying doses of amp B would be most effective in inducing lameness in meat type goats and (2) develop a facial grimace scale for goats. Lameness was produced by an intra-articular injection of amphotericin B into the left hind lateral claw distal interphalangeal joint with either a 5 mg/0.25 mL (high-low, 5 mg of amphotericin B in a volume of 0.25 mL), 5 mg/0.5 mL (high-high, 5 mg of amphotericin B in a volume of 0.5 mL), or a 2.5 mg/0.25 mL (low-low, 2.5 mg of amphotericin B in a volume of 0.25 mL). A saline treatment of 0.5 mL was used as control (0.9% sterile saline solution). Lameness response was analyzed by infrared thermography (IRT) at the induced joint, mechanical-nociception threshold (MNT), visual lameness scoring (VLS), a visual analogue scale (VAS), kinetic gait analysis (KGA), plasma cortisol (CORT), substance P (Sub P), and behavior scoring. The IRT and MNT values differed by timepoint ( P  ≤ 0.0001). Results from VLS showed the HL treatment was the most effective at inducing lameness (6/6 goats became lame compared to HH 4/6 and LL 2/6). At 24, 48, and 72 h, VAS scores were significantly higher when comparing HL to all other treatment groups ( P  = 0.0003). Both behavior observers (1 and 2) reported a significant time effect ( P  = 0.05), with goats exhibiting more facial grimacing at 24 h post-lameness induction. From these data, an optimal dose for a repeatable lameness induction model in goats was aquired. An effective Goat Grimace Scale (GGS) was also developed to evaluate pain responses in goats., Competing Interests: The author and co-authors do not have any conflicts of interest to disclose., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science.)

The objectives of this descriptive study were to (1) describe the pharmacokinetics of salicylic acid (SA) in the milk and plasma of postpartum dairy cattle following oral administration of acetylsalicylic acid (ASA; aspirin), (2) to estimate a recommended milk withdrawal period for dairy cattle treated with ASA, and (3) to determine the effect of ASA administration on plasma prostaglandin E 2 metabolite (PGEM) concentrations. Primiparous (n = 3) and multiparous (n = 7) postpartum Holstein dairy cows received 2 oral treatments with ASA at 200 mg/kg of body weight, 24 h apart. Concentrations of SA in plasma and milk from 0 h through 120 h after ASA administration were analyzed using ultra performance liquid chromatography triple quadrupole mass spectrometry and a milk withdrawal period was estimated using the United States Food and Drug Administration Milk Discard App in R. Two withdrawal periods were estimated: (1) a whole-herd treatment scenario with no dilution factor and (2) an individual animal treatment scenario with a bulk tank factor included in analysis. Plasma PGEM concentrations in samples from 0 h to 24 h after ASA administration were determined using a commercially available competitive ELISA. Milk SA concentrations were undetected in all cows by 48 h after the last ASA treatment. Secondary peaks were observed in plasma at 58 and 82 h after the last treatment and in milk at 87 h after the last treatment. In the absence of a tolerance for SA in milk, the estimated milk withdrawal periods were (1) 156 h for the whole-herd treatment scenario and (2) 120 h for the individual animal treatment scenario. Plasma PGEM concentrations were reduced compared with baseline for up to 12 h after ASA administration, with the greatest reduction observed at 2 h. Results from this study suggest that the current milk withhold recommendation for dairy cattle administered ASA may need revision to 120 h (5 d) and that ASA administration may mitigate postpartum inflammation through reduction in prostaglandin production for up to 12 h after treatment. Pharmacokinetic and milk withdrawal data from this study will inform future recommendations for extra-label use of aspirin in postpartum dairy cows. Further research is required to determine the basis for the secondary SA peaks and to elucidate the long-term effects of ASA administration on dairy cow health., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ~1.7 × 10 7 member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ~4 × 10 3 rRNA variants. We further demonstrate the prevalence of positive epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.

In vitro construction of Escherichia coli ribosomes could elucidate a deeper understanding of these complex molecular machines and make possible the production of synthetic variants with new functions. Toward this goal, we recently developed an integrated synthesis, assembly and translation (iSAT) system that allows for co-activation of ribosomal RNA (rRNA) transcription and ribosome assembly, mRNA transcription and protein translation without intact cells. Here, we discovered that macromolecular crowding and reducing agents increase overall iSAT protein synthesis; the combination of 6% w/v Ficoll 400 and 2 mM DTBA yielded approximately a five-fold increase in overall iSAT protein synthesis activity. By utilizing a fluorescent RNA aptamer, fluorescent reporter proteins and ribosome sedimentation analysis, we showed that crowding agents increase iSAT yields by enhancing translation while reducing agents increase rRNA transcription and ribosome assembly. Finally, we showed that iSAT ribosomes possess ∼70% of the protein synthesis activity of in vivo-assembled E. coli ribosomes. This work improves iSAT protein synthesis through the addition of crowding and reducing agents, provides a thorough understanding of the effect of these additives within the iSAT system and demonstrates how iSAT allows for manipulation and analysis of ribosome biogenesis in the context of an in vitro transcription-translation system., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Complete cell-free synthesis of ribosomes could make possible minimal cell projects and the construction of variant ribosomes with new functions. Recently, we reported the development of an integrated synthesis, assembly, and translation (iSAT) method for in vitro construction of Escherichia coli ribosomes. iSAT allows simultaneous rRNA synthesis, ribosome assembly, and reporter protein expression as a measure of ribosome activity. Here, we explore causes of iSAT reaction termination to improve efficiency and yields. We discovered that phosphoenolpyruvate (PEP), the secondary energy substrate, and nucleoside triphosphates (NTPs) were rapidly degraded during iSAT reactions. In turn, we observed a significant drop in the adenylate energy charge and termination of protein synthesis. Furthermore, we identified that the accumulation of inorganic phosphate is inhibitory to iSAT. Fed-batch replenishment of PEP and magnesium glutamate (to offset the inhibitory effects of accumulating phosphate by repeated additions of PEP) prior to energy depletion prolonged the reaction duration 2-fold and increased superfolder green fluorescent protein (sfGFP) yield by ~75%. By adopting a semi-continuous method, where passive diffusion enables substrate replenishment and byproduct removal, we prolonged iSAT reaction duration 5-fold and increased sfGFP yield 7-fold to 7.5 ± 0.7 μmol L(-1). This protein yield is the highest ever reported for iSAT reactions. Our results underscore the critical role energy substrates play in iSAT and highlight the importance of understanding metabolic processes that influence substrate depletion for cell-free synthetic biology.

In vitro ribosome construction could enable studies of ribosome assembly and function, provide a route toward constructing minimal cells for synthetic biology, and permit the construction of ribosome variants with new functions. Toward these long-term goals, we recently reported on an integrated, one-pot ribosomal RNA synthesis (rRNA), ribosome assembly, and translation technology (termed iSAT) for the construction of Escherichia coli ribosomes in crude ribosome-free S150 extracts. Here, we aimed to improve the activity of iSAT through transcriptional tuning. Specifically, we increased transcriptional efficiency through 3' modifications to the rRNA gene sequences, optimized plasmid and polymerase concentrations, and demonstrated the use of a T7-promoted rRNA operon for stoichiometrically balanced rRNA synthesis and native rRNA processing. Our modifications produced a 45-fold improvement in iSAT protein synthesis activity, enabling synthesis of 429 ± 15 nmol/l green fluorescent protein in 6 h batch reactions. Further, we show that the translational activity of ribosomes purified from iSAT reactions is about 20% the activity of native ribosomes purified directly from E. coli cells. Looking forward, we believe iSAT will enable unique studies to unravel the systems biology of ribosome biogenesis and open the way to new methods for making and studying ribosomal variants., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties.

Modification of microRNA sequences by the 3' addition of nucleotides to generate so-called "isomiRs" adds to the complexity of miRNA function, with recent reports showing that 3' modifications can influence miRNA stability and efficiency of target repression. Here, we show that the 3' modification of miRNAs is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species ranging from C. elegans to human. The modifications result predominantly from adenylation and uridylation and are seen across tissue types, disease states, and developmental stages. To quantitatively profile 3' nucleotide additions, we developed and validated a novel assay based on NanoString Technologies' nCounter platform. For certain miRNAs, the frequency of modification was altered by processes such as cell differentiation, indicating that 3' modification is a biologically regulated process. To investigate the mechanism of 3' nucleotide additions, we used RNA interference to screen a panel of eight candidate miRNA nucleotidyl transferases for 3' miRNA modification activity in human cells. Multiple enzymes, including MTPAP, PAPD4, PAPD5, ZCCHC6, ZCCHC11, and TUT1, were found to govern 3' nucleotide addition to miRNAs in a miRNA-specific manner. Three of these enzymes-MTPAP, ZCCHC6, and TUT1-have not previously been known to modify miRNAs. Collectively, our results indicate that 3' modification observed in next-generation small RNA sequencing data is a biologically relevant process, and identify enzymatic mechanisms that may lead to new approaches for modulating miRNA activity in vivo.

Synthetic biology is a nascent technical discipline that seeks to enable the design and construction of novel biological systems to meet pressing societal needs. However, engineering biology still requires much trial and error because we lack effective approaches for connecting basic "parts" into higher-order networks that behave as predicted. Developing strategies for improving the performance and sophistication of our designs is informed by two overarching perspectives: "bottom-up" and "top-down" considerations. Using this framework, we describe a conceptual model for developing novel biological systems that function and interact with existing biological components in a predictable fashion. We discuss this model in the context of three topical areas: biochemical transformations, cellular devices and therapeutics, and approaches that expand the chemistry of life. Ten years after the construction of synthetic biology's first devices, the drive to look beyond what does exist to what can exist is ushering in an era of biology by design.

Polymorphisms at 8q24 are robustly associated with prostate cancer risk. The risk variants are located in nonprotein coding regions and their mechanism has not been fully elucidated. To further dissect the function of this locus, we tested two hypotheses: (a) unannotated microRNAs (miRNA) are transcribed in the region, and (b) this region is a cis-acting enhancer. Using next generation sequencing, 8q24 risk regions were interrogated for known and novel miRNAs in histologically normal radical prostatectomy tissue. We also evaluated the association between the risk variants and transcript levels of multiple genes, focusing on the proto-oncogene, MYC. RNA expression was measured in histologically normal and tumor tissue from 280 prostatectomy specimens (from 234 European American and 46 African American patients), and paired germline DNA from each individual was genotyped for six 8q24 risk single nucleotide polymorphisms. No evidence was found for significant miRNA transcription within 8q24 prostate cancer risk loci. Likewise, no convincing association between RNA expression and risk allele status was detected in either histologically normal or tumor tissue. To our knowledge, this is one of the first and largest studies to directly assess miRNA in this region and to systematically measure MYC expression levels in prostate tissue in relation to inherited risk variants. These data will help to direct the future study of this risk locus.

Background: MicroRNAs (miRNAs) are small regulatory RNAs that are implicated in cancer pathogenesis and have recently shown promise as blood-based biomarkers for cancer detection. Epithelial ovarian cancer is a deadly disease for which improved outcomes could be achieved by successful early detection and enhanced understanding of molecular pathogenesis that leads to improved therapies. A critical step toward these goals is to establish a comprehensive view of miRNAs expressed in epithelial ovarian cancer tissues as well as in normal ovarian surface epithelial cells., Methodology: We used massively parallel pyrosequencing (i.e., "454 sequencing") to discover and characterize novel and known miRNAs expressed in primary cultures of normal human ovarian surface epithelium (HOSE) and in tissue from three of the most common histotypes of ovarian cancer. Deep sequencing of small RNA cDNA libraries derived from normal HOSE and ovarian cancer samples yielded a total of 738,710 high-quality sequence reads, generating comprehensive digital profiles of miRNA expression. Expression profiles for 498 previously annotated miRNAs were delineated and we discovered six novel miRNAs and 39 candidate miRNAs. A set of 124 miRNAs was differentially expressed in normal versus cancer samples and 38 miRNAs were differentially expressed across histologic subtypes of ovarian cancer. Taqman qRT-PCR performed on a subset of miRNAs confirmed results of the sequencing-based study., Conclusions: This report expands the body of miRNAs known to be expressed in epithelial ovarian cancer and provides a useful resource for future studies of the role of miRNAs in the pathogenesis and early detection of ovarian cancer.

We used massively parallel pyrosequencing to discover and characterize microRNAs (miRNAs) expressed in human embryonic stem cells (hESC). Sequencing of small RNA cDNA libraries derived from undifferentiated hESC and from isogenic differentiating cultures yielded a total of 425,505 high-quality sequence reads. A custom data analysis pipeline delineated expression profiles for 191 previously annotated miRNAs, 13 novel miRNAs, and 56 candidate miRNAs. Further characterization of a subset of the novel miRNAs in Dicer-knockdown hESC demonstrated Dicer-dependent expression, providing additional validation of our results. A set of 14 miRNAs (9 known and 5 novel) was noted to be expressed in undifferentiated hESC and then strongly downregulated with differentiation. Functional annotation analysis of predicted targets of these miRNAs and comparison with a null model using non-hESC-expressed miRNAs identified statistically enriched functional categories, including chromatin remodeling and lineage-specific differentiation annotations. Finally, integration of our data with genome-wide chromatin immunoprecipitation data on OCT4, SOX2, and NANOG binding sites implicates these transcription factors in the regulation of nine of the novel/candidate miRNAs identified here. Comparison of our results with those of recent deep sequencing studies in mouse and human ESC shows that most of the novel/candidate miRNAs found here were not identified in the other studies. The data indicate that hESC express a larger complement of miRNAs than previously appreciated, and they provide a resource for additional studies of miRNA regulation of hESC physiology. Disclosure of potential conflicts of interest is found at the end of this article.

Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small ( approximately 22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.

MicroRNAs are small, non-coding RNAs that influence gene regulatory networks by post-transcriptional regulation of specific messenger RNA targets. MicroRNA expression is dysregulated in human malignancies, frequently leading to loss of expression of certain microRNAs. We report that expression of hsa-miR-342, a microRNA encoded in an intron of the gene EVL, is commonly suppressed in human colorectal cancer. The expression of hsa-miR-342 is coordinated with that of EVL and our results indicate that the mechanism of silencing is CpG island methylation upstream of EVL. We found methylation at the EVL/hsa-miR-342 locus in 86% of colorectal adenocarcinomas and in 67% of adenomas, indicating that it is an early event in colorectal carcinogenesis. In addition, we observed a higher frequency of methylation (56%) in histologically normal colorectal mucosa from individuals with concurrent cancer compared to mucosa from individuals without colorectal cancer (12%), suggesting the existence of a 'field defect' involving methylated EVL/hsa-miR-342. Furthermore, reconstitution of hsa-miR-342 in the colorectal cancer cell line HT-29 induced apoptosis, suggesting that this microRNA could function as a proapoptotic tumor suppressor. In aggregate, these results support a novel mechanism for silencing intronic microRNAs in cancer by epigenetic alterations of cognate host genes.

Activation of the Xenopus bone morphogenetic protein (BMP) pathway is coincident with the onset of zygotic transcription but requires maternal signaling proteins. The mechanisms controlling the translation of mRNAs that encode proteins of the BMP pathway were investigated by using polysome association as an assay for translational activity. Our results indicate that five different mRNAs encoding proteins of the BMP pathway were translationally regulated during Xenopus development. These mRNAs were either not associated or inefficiently associated with polysomes in oocytes, and each was recruited to polysomes at a different developmental stage. The Smad1 and ALK-2 mRNAs were recruited to polysomes during oocyte maturation, whereas the BMP-7 and XSTK9 mRNAs were recruited during the early stages of embryogenesis. The ALK-3 mRNA was not efficiently associated with polysomes during any maternal stage of development and was efficiently recruited to polysomes only after the onset of zygotic transcription. In general, for all stages except oocytes, polysome recruitment was associated with the presence of a 3' poly(A) tail. However, there was not an obvious correlation between the absolute length of poly(A) and the efficiency of polysome recruitment, indicating that the relationship between poly(A) tail length and translation during early frog embryogenesis is complex. We further focused on the BMP-7 mRNA and demonstrated that sequence elements within the 3'UTR were necessary for recruitment of the BMP-7 mRNA to polysomes and sufficient to direct the addition of poly(A) and activate translation of a reporter during embryogenesis. Interestingly, the BMP-7 mRNA lacks the previously defined eCPE sequences proposed to direct poly(A) addition and translational activation during embryogenesis. The implications of our findings for translational regulation of maternal mRNAs during embryogenesis and for the activation of the BMP pathway are discussed., (Copyright 2001 Academic Press.)