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Recent cross-fertilisation among the fields of critical legal geography, political ecology and environmental ethics has created opportunities to examine new legal designations for more-than-human entities such as rivers. In particular, theorisations of how more-than-human assemblages ontologically co-constitute the law challenge interpretations of legal structures as purely human creations and encourage scholars to examine manifestations of non-anthropocentrism, which is defined here as an ethical position that elevates non-humans’ moral standing to that of humans. This article adopts an historical case study approach to examine an environmental conflict that occurred in the 1970s involving non-anthropocentrism and Montana water law. Specifically, this article draws from critical legal geography’s understanding of the law and the environment as being co-constituted to argue that both elements of a non-anthropocentric environmental ethic and the influence of Yellowstone River as a more-than-human entity shaped Montana Fish and Game’s position during the Yellowstone River Basin water reservation process. This article further argues that the combination of these influences affected legal interpretation of the 1973 Montana Water Use Act’s 'minimum stream flow' text and helped reconstitute the Act to include non-anthropocentric elements.

Abstract Providing an anodic potential in a bio‐electrochemical system to the obligate aerobe Pseudomonas putida enables anaerobic survival and allows the cells to overcome redox imbalances. In this setup, the bacteria could be exploited to produce chemicals via oxidative pathways at high yield. However, the absence of anaerobic growth and low carbon turnover rates remain as obstacles for the application of such an electro‐fermentation technology. Growth and carbon turnover start with carbon uptake into the periplasm and cytosol. P. putida KT2440 has three native transporting systems for glucose, each differing in energy and redox demand. This architecture previously led to the hypothesis that internal redox and energy constraints ultimately limit cytoplasmic carbon utilization in a bio‐electrochemical system. However, it remains largely unclear which uptake route is predominantly used by P. putida under electro‐fermentative conditions. To elucidate this, we created three gene deletion mutants of P. putida KT2440, forcing the cells to exclusively utilize one of the routes. When grown in a bio‐electrochemical system, the pathway mutants were heavily affected in terms of sugar consumption, current output and product formation. Surprisingly, however, we found that about half of the acetate formed in the cytoplasm originated from carbon that was put into the system via the inoculation biomass, while the other half came from the consumption of substrate. The deletion of individual sugar uptake routes did not alter significantly the secreted acetate concentrations among different strains even with different carbon sources. This means that the stoichiometry of the sugar uptake routes is not a limiting factor during electro‐fermentation and that the low rates might be caused by other reasons, for example energy limitations or a yet‐to‐be‐identified oxygen‐dependent regulatory mechanism.

Can contemporary cognitive science explain clinical expertise? We argue that the answer could be “no.” In support of this, we provide an analysis of two of the most essential expressions of clinical expertise in nursing and medicine, the ability to run a code blue and the ability to diagnose congestive heart failure. We show how it makes sense to treat both as examples of what we call inference to the best action, and we then argue that two of the standard explanatory paradigms of cognitive science — the Humean and Bayesian paradigms — are unable to provide a plausible analysis of inference to the best action.

ABSTRACT Thymidylate kinases (TMPKs) play an essential role in DNA biosynthesis across all domains of life by catalyzing dTMP phosphorylation to dTDP. In Pseudomonas putida KT2440, a model Gram-negative soil bacterium, tmk is disrupted by a 65-kb genomic island (GI), posing questions about the origin of the essential TMPK function. To solve this long-standing evolutionary riddle, we addressed three competing hypotheses: (i) assembly of two Tmk segments into a functional protein, (ii) complementation by a deoxynucleotide monophosphate kinase encoded within the GI, or (iii) fulfillment of the essential function by the product of PP_3363, yet another gene annotated as “thymidylate kinase.” Systematic genome engineering, quantitative physiology and targeted proteomics, complementation assays, phylogenetic analysis, and structure homology modeling were combined to investigate the role of genes within the GI. Our findings revealed that the GI-encoded dNMPK gene PP_1964 plays a critical role in complementing the disrupted TMPK function—exposing a non-essential character for the native PP_3363 gene and the tmk pseudogene. This dNMPK was found to be structurally related to that of bacteriophage T4, as part of a distinct evolutionary domain connected to mobile genetic elements and phages. The recursive genome reduction approach in this work deepens our understanding of the genetic architecture of a model bacterium while it provides evidence that the essential TMPK function has been acquired by horizontal gene transfer. Furthermore, the insights gained in the present study have broader implications for understanding the essentiality and functionality of dNMPK homologs in other bacteria. IMPORTANCE Investigating fundamental aspects of metabolism is vital for advancing our understanding of the diverse biochemical capabilities and biotechnological applications of bacteria. The origin of the essential thymidylate kinase function in the model bacterium Pseudomonas putida KT2440, seemingly interrupted due to the presence of a large genomic island that disrupts the cognate gene, eluded a satisfactory explanation thus far. This is a first-case example of an essential metabolic function, likely acquired by horizontal gene transfer, which “landed” in a locus encoding the same activity. As such, foreign DNA encoding an essential dNMPK could immediately adjust to the recipient host—instead of long-term accommodation and adaptation. Understanding how these functions evolve is a major biological question, and the work presented here is a decisive step toward this direction. Furthermore, identifying essential and accessory genes facilitates removing those deemed irrelevant in industrial settings—yielding genome-reduced cell factories with enhanced properties and genetic stability.

The accretion/ejection processes in T Tauri stars are fundamental to their physical evolution, while also impacting the properties and evolution of the circumstellar material at a time when planet formation takes place. To date, the characterization of ongoing accretion processes in stellar pairs at 5–50 au scales has been challenging as high-angular resolution spectrographs are required to extract the spectral features of each component. We present the analysis of spectroscopic observations of the tight (160 mas, 25 au) T Tauri system HT Lup A/B, obtained with MUSE at the Very Large Telescope in 2021 March and July. We focus on constraining the accretion/ejection processes and variability of the secondary component HT Lup B by searching for accretion tracers by applying high-resolution spectral differential imaging techniques. We retrieve strong (signal-to-noise ratio > 5) H α , H β, and [O i ] λ 6300 emission in both epochs. The H α and H β line fluxes showcase high variability, with variations up to 200%–300% between epochs. The fluxes are consistent with accretion rates of 3× 10 ^−9 M _⊙ yr ^−1 and 8 × 10 ^−10 M _⊙ yr ^−1 for the first and second epochs, respectively. We attribute the increased accretion activity during the first night to a “burst-like” event, followed by a relaxation period more representative of the common accretion activity of the system. The [O i ] λ 6300 line profiles remain relatively similar between epochs and suggest ejection rates on the order of 10 ^−9 −10 ^−10 M _⊙ yr ^−1 , compatible with moderate disk wind emission. Our results also indicate that the accretion processes of HT Lup B are compatible with Classical T Tauri stars, unlike previous classifications.

Summary The potential of LacI/Ptrc, XylS/Pm, AlkS/PalkB, CprK/PDB3 and ChnR/PchnB regulatory nodes, recruited from both Gram‐negative and Gram‐positive bacteria, as the source of parts for formatting expression cargoes following the Standard European Vector Architecture (SEVA) has been examined. The five expression devices, which cover most known regulatory configurations in bacteria were assembled within exactly the same plasmid backbone and bearing the different functional segments arrayed in an invariable DNA scaffold. Their performance was then analysed in an Escherichia coli strain of reference through the readout of a fluorescence reporter gene that contained strictly identical translation signal elements. This approach allowed us to describe and compare the cognate expression systems with quantitative detail. The constructs under scrutiny diverged considerably in their capacity, expression noise, inducibility and ON/OFF ratios. Inspection of such a variance exposed the different constraints that rule the optimal arrangement of functional DNA segments in each case. The data highlighted also the ease of standardizing inducer‐responsive devices subject to transcriptional activation as compared to counterparts based on repressors. The study resulted in a defined collection of formatted expression cargoes lacking any cross talk while offering a panoply of choices to potential users and help interoperability of the specific constructs.

ABSTRACT The soil bacterium Pseudomonas putida is a robust biomanufacturing host that assimilates a broad range of substrates while efficiently coping with adverse environmental conditions. P. putida is equipped with functions related to one-carbon (C1) compounds (e.g. methanol, formaldehyde, and formate) oxidation—yet pathways to assimilate these carbon sources are largely absent. In this work, we adopted a systems-level approach to study the genetic and molecular basis of C1 metabolism in P. putida. RNA sequencing identified two oxidoreductases, encoded by PP_0256 and PP_4596, transcriptionally active in the presence of formate. Quantitative physiology of deletion mutants revealed growth defects at high formate concentrations, pointing to an important role of these oxidoreductases in C1 tolerance. Moreover, we describe a concerted detoxification process for methanol and formaldehyde, the C1 intermediates upstream formate. Alcohol oxidation to highly-reactive formaldehyde by PedEH and other broad-substrate-range dehydrogenases underpinned the (apparent) suboptimal methanol tolerance of P. putida. Formaldehyde was mostly processed by a glutathione-dependent mechanism encoded in the frmAC operon, and thiol-independent FdhAB and AldB-II overtook detoxification at high aldehyde concentrations. Deletion strains were constructed and characterized towards unveiling these biochemical mechanisms, underscoring the worth of P. putida for emergent biotechnological applications—e.g. engineering synthetic formatotrophy and methylotrophy. IMPORTANCE C1 substrates continue to attract interest in biotechnology, as their use is both cost-effective and ultimately expected to mitigate the impact of greenhouse gas emissions. However, our current understanding of bacterial C1 metabolism remains relatively limited in species that cannot grow on (i.e., assimilate) these substrates. Pseudomonas putida, a model Gram-negative environmental bacterium, constitutes a prime example of this sort. The biochemical pathways active in response to methanol, formaldehyde, and formate have been largely overlooked—although the ability of P. putida to process C1 molecules has been previously alluded to in the literature. By using a systems-level strategy, this study bridges such knowledge gap through the identification and characterization of mechanisms underlying methanol, formaldehyde, and formate detoxification—including hitherto unknown enzymes that act on these substrates. The results reported herein both expand our understanding of microbial metabolism and lay a solid foundation for engineering efforts toward valorizing C1 feedstocks.

Abstract The domestication of plants has commonly resulted in the loss of plant defense metabolites, with important consequences for the plants' interactions with herbivores and their natural enemies. Squash domestication started 10′000 years ago and has led to the loss of cucurbitacins, which are highly toxic triterpenes. The banded cucumber beetle (Diabrotica balteata), a generalist herbivore, is adapted to feed on plants from the Cucurbitaceae and is known to sequester cucurbitacins, supposedly for its own defense. However, the evidence for this is inconclusive. In this study we tested the impact of squash domestication on the chemical protection of D. balteata larvae against a predatory rove beetle (Dalotia coriaria). We found that cucurbitacins do not defend the larvae against this common soil dwelling predator. In fact, D. balteata larvae were less attacked when they fed on cucurbitacin‐free roots of domesticated varieties compared to high‐cucurbitacin roots of wild plants. This study appears to be the first to look at the consequences of plant domestication on belowground tritrophic interactions. Our results challenge the generalized assumption that sequestered cucurbitacins protect this herbivore against natural enemies, and instead reveals an opposite effect that may be due to a tradeoff between coping with cucurbitacins and avoiding predation.

Abstract Aotearoa New Zealand is the last major landmass settled by people, and therefore provides a recent record of ecological legacy effects in the coastal zone. Large‐scale land clearances of forests accelerated over the last century, affecting the concentration of suspended sediments, light environment, and nutrient composition on rocky reefs, and consequently the distribution, abundance, and composition of algal forests. The environmental effects were compounded in many places by overfishing and long‐term declines of large predatory species, often leading to proliferation and extensive grazing by sea urchins. In this study, we examine these processes in three biogeographic regions that have been differentially affected by ecological legacy effects. The study was based on the depth‐specific associations between sea urchins (Evechinus chloroticus) and the common kelp (Ecklonia radiata) from multiple sites within each region, some of which were sampled over two decades within no‐take marine reserves and in actively fished areas. Satellite‐derived estimates of water‐column properties were used as proxies for the relative effects of coastal sedimentation on kelp forest habitats. We then used an information‐theoretic framework to assess the relative factor weightings of marine reserve effects on urchin density, total suspended solids (TSS), and regional urchin density on the depth‐specific density of Ecklonia radiata. The fishing effects were significant within and outside of reserves in the northern and central regions, but the effect sizes were by far strongest in the northern region. In the central region characterized by extensive land clearance and forestry combined with high coastal retention of water, the concentration of TSS had a major influence on the depth distribution of kelp, confining it to shallow water (

Abstract The development of chemistry is reported to implement selective dual‐wavelength olefin metathesis polymerization for continuous additive manufacturing (AM). A resin formulation based on dicyclopentadiene is produced using a latent olefin metathesis catalyst, various photosensitizers (PSs) and photobase generators (PBGs) to achieve efficient initiation at one wavelength (e.g., blue light) and fast catalyst decomposition and polymerization deactivation at a second (e.g., UV‐light). This process enables 2D stereolithographic (SLA) printing, either using photomasks or patterned, collimated light. Importantly, the same process is readily adapted for 3D continuous AM, with printing rates of 36 mm h–1 for patterned light and up to 180 mm h–1 using un‐patterned, high intensity light.

Summary Pseudomonas species have become reliable platforms for bioproduction due to their capability to tolerate harsh conditions imposed by large‐scale bioprocesses and their remarkable resistance to diverse physicochemical stresses. The last few years have brought forth a variety of synthetic biology tools for the genetic manipulation of pseudomonads, but most of them are either applicable only to obtain certain types of mutations, lack efficiency, or are not easily accessible to be used in different Pseudomonas species (e.g. natural isolates). In this work, we describe a versatile, robust and user‐friendly procedure that facilitates virtually any kind of genomic manipulation in Pseudomonas species in 3–5 days. The protocol presented here is based on DNA recombination forced by double‐stranded DNA cuts (through the activity of the I‐SceI homing meganuclease from yeast) followed by highly efficient counterselection of mutants (aided by a synthetic CRISPR‐Cas9 device). The individual parts of the genome engineering toolbox, tailored for knocking genes in and out, have been standardized to enable portability and easy exchange of functional gene modules as needed. The applicability of the procedure is illustrated both by eliminating selected genomic regions in the platform strain P. putida KT2440 (including difficult‐to‐delete genes) and by integrating different reporter genes (comprising novel variants of fluorescent proteins) into a defined landing site in the target chromosome.

Precise genome engineering has become a commonplace technique for metabolic engineering. Also, insertion, deletion and alteration of genes and other functional DNA sequences are essential for understanding and engineering cells. Several techniques have been developed to this end (e.g., CRISPR/Cas-assisted methods, homologous recombination, or λ Red recombineering), yet most of them rely on the use of auxiliary plasmids, which have to be cured after the editing procedure. Temperature-sensitive replicons, counter-selectable markers or repeated passaging of plasmid-bearing cells have been traditionally employed to circumvent this hurdle. While these protocols work reasonably well in some bacteria, they are not applicable for other species or are time consuming and laborious. Here, we present a fast and versatile protocol of fluorescent marker-assisted genome editing in Pseudomonas putida, followed by clean curing of auxiliary plasmids through user-controlled plasmid replication. One fluorescent marker facilitates identification of genome-edited colonies, while the second reporter enables detection of plasmid-free bacterial clones. Not only is this protocol the fastest available for Pseudomonas species, but it can be easily adapted to any type of genome modifications, including sequence deletions, insertions, and replacements.Graphical abstract:Rapid genome engineering of Pseudomonas with curable plasmids

Genome engineering of non-conventional microorganisms calls for the development of dedicated synthetic biology tools. Pseudomonas putida is a Gram-negative, non-pathogenic soil bacterium widely used for metabolic engineering owing to its versatile metabolism and high levels of tolerance to different types of stress. Genome editing of P. putida largely relies on homologous recombination events, assisted by helper plasmid-based expression of genes encoding DNA modifying enzymes. Plasmid curing from selected isolates is the most tedious and time-consuming step of this procedure, and implementing commonly used methods to this end in P. putida (e.g. temperature-sensitive replicons) is often impractical. To tackle this issue, we have developed a toolbox for both target- and self-curing of plasmid DNA in Pseudomonas species. Our method enables plasmid-curing in a simple cultivation step by combining in vivo digestion of vectors by the I-SceI homing nuclease with synthetic control of plasmid replication, triggered by the addition of a cheap chemical inducer (3-methylbenzoate) to the medium. The system displays an efficiency of vector curing >90% and the screening of plasmid-free clones is greatly facilitated by the use of fluorescent markers that can be selected according to the application intended. Furthermore, quick genome engineering of P. putida using self-curing plasmids is demonstrated through genome reduction of the platform strain EM42 by eliminating all genes encoding β-lactamases, the catabolic ben gene cluster, and the pyoverdine synthesis machinery. Physiological characterization of the resulting streamlined strain, P. putida SEM10, revealed advantageous features that could be exploited for metabolic engineering.

Summary: The p53 tumor suppressor pathway is frequently inactivated in human cancers. However, there are some cancer types without commonly recognized alterations in p53 signaling. Here we report that histone demethylase KDM5A is involved in the regulation of p53 activity. KDM5A is significantly amplified in multiple types of cancers, an event that tends to be mutually exclusive to p53 mutation. We show that KDM5A acts as a negative regulator of p53 signaling through inhibition of p53 translation via suppression of a subgroup of eukaryotic translation initiation genes. Genetic deletion of KDM5A results in upregulation of p53 in multiple lineages of cancer cells and inhibits tumor growth in a p53-dependent manner. In addition, we have identified a regulatory loop between p53, miR-34, and KDM5A, whereby the induction of miR-34 leads to suppression of KDM5A. Thus, our findings reveal a mechanism by which KDM5A inhibits p53 translation to modulate cancer progression. : Molecular Mechanism of Gene Regulation; Cancer Subject Areas: Molecular Mechanism of Gene Regulation, Cancer

Several techniques are available to characterize acoustic liners when subject to grazing flow and high sound pressure level (SPL). Although the in situ technique started as the primary experimental procedure, impedance eduction techniques have gained popularity over the past years. However, there is a lack of comparison between these group of methods, especially at conditions typically found in turbofan engines. In this work, in situ and impedance eduction techniques are compared at high flow velocities and SPL using typical acoustic liner test samples and considering uniform flow. Both upstream and downstream acoustic wave propagation will also be considered in view of the discrepancies recently observed by eduction methods. A new method to compensate the instrumentation effect in the in situ technique is proposed and validated. Results are obtained for bulk Mach numbers up to 0.5 and SPLs up to 145 dB for both in situ and two eduction techniques. The three methods presents good agreement in the absence of flow. Unexpected results are observed with higher flow Mach numbers using the eduction technique. [ABSTRACT FROM AUTHOR]

Recent cross-fertilisation among the fields of critical legal geography, political ecology and environmental ethics has created opportunities to examine new legal designations for more-than-human entities such as rivers. In particular, theorisations of how more-than-human assemblages ontologically co-constitute the law challenge interpretations of legal structures as purely human creations and encourage scholars to examine manifestations of non-anthropocentrism, which is defined here as an ethical position that elevates non-humans' moral standing to that of humans. This article adopts an historical case study approach to examine an environmental conflict that occurred in the 1970s involving non-anthropocentrism and Montana water law. Specifically, this article draws from critical legal geography's understanding of the law and the environment as being co-constituted to argue that both elements of a non-anthropocentric environmental ethic and the influence of Yellowstone River as a more-than-human entity shaped Montana Fish and Game's position during the Yellowstone River Basin water reservation process. This article further argues that the combination of these influences affected legal interpretation of the 1973 Montana Water Use Act's 'minimum stream flow' text and helped reconstitute the Act to include non-anthropocentric elements. [ABSTRACT FROM AUTHOR]

Providing an anodic potential in a bio‐electrochemical system to the obligate aerobe Pseudomonas putida enables anaerobic survival and allows the cells to overcome redox imbalances. In this setup, the bacteria could be exploited to produce chemicals via oxidative pathways at high yield. However, the absence of anaerobic growth and low carbon turnover rates remain as obstacles for the application of such an electro‐fermentation technology. Growth and carbon turnover start with carbon uptake into the periplasm and cytosol. P. putida KT2440 has three native transporting systems for glucose, each differing in energy and redox demand. This architecture previously led to the hypothesis that internal redox and energy constraints ultimately limit cytoplasmic carbon utilization in a bio‐electrochemical system. However, it remains largely unclear which uptake route is predominantly used by P. putida under electro‐fermentative conditions. To elucidate this, we created three gene deletion mutants of P. putida KT2440, forcing the cells to exclusively utilize one of the routes. When grown in a bio‐electrochemical system, the pathway mutants were heavily affected in terms of sugar consumption, current output and product formation. Surprisingly, however, we found that about half of the acetate formed in the cytoplasm originated from carbon that was put into the system via the inoculation biomass, while the other half came from the consumption of substrate. The deletion of individual sugar uptake routes did not alter significantly the secreted acetate concentrations among different strains even with different carbon sources. This means that the stoichiometry of the sugar uptake routes is not a limiting factor during electro‐fermentation and that the low rates might be caused by other reasons, for example energy limitations or a yet‐to‐be‐identified oxygen‐dependent regulatory mechanism. [ABSTRACT FROM AUTHOR]

INTRODUCTION:Whereas scientists depend on the language of probability to relay information about hazards, risk communication may be more effective when embedding scientific information in narratives. The persuasive power of narratives is theorized to reside, in part, in narrative transportation. PURPOSE:This study seeks to advance the science of stories in risk communication by measuring real-time affective responses as a proxy indicator for narrative transportation during science messages that present scientific information in the context of narrative. METHODS:This study employed a within-subjects design in which participants (n = 90) were exposed to eight science messages regarding flood risk. Conventional science messages using probability and certainty language represented two conditions. The remaining six conditions were narrative science messages that embedded the two conventional science messages within three story forms that manipulated the narrative mechanism of character selection. Informed by the Narrative Policy Framework, the characters portrayed in the narrative science messages were hero, victim, and victim-to-hero. Natural language processing techniques were applied to identify and rank hero and victim vocabularies from 45 resident interviews conducted in the study area; the resulting classified vocabulary was used to build each of the three story types. Affective response data were collected over 12 group sessions across three flood-prone communities in Montana. Dial response technology was used to capture continuous, second-by-second recording of participants' affective responses while listening to each of the eight science messages. Message order was randomized across sessions. ANOVA and three linear mixed-effects models were estimated to test our predictions. RESULTS:First, both probabilistic and certainty science language evoked negative affective responses with no statistical differences between them. Second, narrative science messages were associated with greater variance in affective responses than conventional science messages. Third, when characters are in action, variation in the narrative mechanism of character selection leads to significantly different affective responses. Hero and victim-to-hero characters elicit positive affective responses, while victim characters produce a slightly negative response. CONCLUSIONS:In risk communication, characters matter in audience experience of narrative transportation as measured by affective responses.

Although the literature extensively covers the development of battery chargers control strategies, a comparison of these strategies remains a literary gap. The inherent conditions (i.e., State of Health and State of Charge) of each unit in the Battery Energy Storage Systems directly influence the charger control techniques for extending battery lifetime, which makes modular battery chargers an appealing topology for this analysis. This work groups charger control strategies presented in the literature into two: Adapted SoC strategies, directly linked to the field of overstress management, and SoH strategies, which are directly linked to the field of wear-out management. The methodology for comparing the control strategies encompasses battery lifetime, charger, and photovoltaic plant models. Three distinct cases were simulated using real measure data from a solar power plant and a battery model provided by MathWorks®. The results evidence that the Capacity Fade and Energy Throughput strongly depend on the strategy. The controller action evidences the previous statement, as the strategies have different goals that are related to each field. Furthermore, this work analyses the effect of the estimation process in the action of the controller.

The electrowetting-induced detachment of droplets from solid surfaces is important for numerous applications in the fields of heat transfer and fluid mechanics. The forced oscillations of droplets on solid surfaces and their ability to detach are studied. In this study, the process is efficiently simulated by implementing a powerful methodology developed by our team. Our results agree with experiments showing that optimal detachment, in terms of actuation energy, is achieved when the application of voltage is synchronized with the spreading time of the droplet. Under these conditions, the droplet oscillates with a period close to that of a mirrored Rayleigh droplet. The relationship between the droplet's oscillation period and its physical properties is examined. During voltage-droplet synchronization, the droplet's ability to detach depends mostly on its contact angle, its viscosity, and the applied voltage. An energy analysis is also conducted, revealing how energy is supplied to the droplet by electrowetting-induced detachment. [ABSTRACT FROM AUTHOR]

Instrument fidelity in message testing research hinges upon how precisely messages operationalize treatment conditions. However, numerous message testing studies have unmitigated threats to validity and reliability because no established procedures exist to guide construction of message treatments. Their construction typically occurs in a black box, resulting in suspect inferential conclusions about treatment effects. Because a mixed methods approach is needed to enhance instrument fidelity in message testing research, this article contributes to the field of mixed methods research by presenting an integrated multistage procedure for constructing precise message treatments using an exploratory sequential mixed methods design. This work harnesses the power of integration through crossover analysis to improve instrument fidelity in message testing research through the use of natural language processing (NLP). [ABSTRACT FROM AUTHOR]