VPg-based bidirectional synthetic mRNA circuits enable orthogonal protein regulation for high-resolution cell separation.

Synthetic mRNA circuits commonly sense input to produce binary output signals for cell separation. Based on virus-origin cap-independent translation initiation machinery and RBP-aptamer interaction, we designed smart synthetic mRNA-based circuits that sense single input molecules to bidirectionally tune output signals in an orthogonal manner, enabling high-resolution separation of cell populations.

Multiplex miRNA reporting platform for real-time profiling of living cells.

Accurately characterizing cell types within complex cell structures provides invaluable information for comprehending the cellular status during biological processes. In this study, we have developed an miRNA-switch cocktail platform capable of reporting and tracking the activities of multiple miRNAs (microRNAs) at the single-cell level, while minimizing disruption to the cell culture. Drawing on the principles of traditional miRNA-sensing mRNA switches, our platform incorporates subcellular tags and employs intelligent engineering to segment three subcellular regions using two fluorescent proteins. These designs enable the quantification of multiple miRNAs within the same cell. Through our experiments, we have demonstrated the platform's ability to track marker miRNA levels during cell differentiation and provide spatial information of heterogeneity on outlier cells exhibiting extreme miRNA levels. Importantly, this platform offers real-time and in situ miRNA reporting, allowing for multidimensional evaluation of cell profile and paving the way for a comprehensive understanding of cellular events during biological processes.

Improving DNA vaccination performance through a new microbubble design and an optimized sonoporation protocol.

As a non-viral transfection method, ultrasound and microbubble-induced sonoporation can achieve spatially targeted gene delivery with synergistic immunostimulatory effects. Here, we report for the first time the application of sonoporation for improving DNA vaccination performance. This study developed a new microbubble design with nanoscale DNA/PEI complexes loaded onto cationic microbubbles to attain significant increases in DNA-loading capacity (0.25 pg per microbubble) and in vitro transfection efficiency. Using live-cell imaging, we revealed the membrane perforation and cellular delivery characteristics of sonoporation. Using luciferase reporter gene for in vivo transfection, we showed that sonoporation increased the transfection efficiency by 40.9-fold when compared with intramuscular injection. Moreover, we comprehensively optimized the sonoporation protocol and further increased the transfection efficiency by 43.6-fold. Immunofluorescent staining results showed that sonoporation effectively activated the MHC-II+ immune cells. Using a hepatitis B DNA vaccine, sonoporation induced significantly higher serum antibody levels when compared with intramuscular injection, and the antibodies sustained for 56 weeks. In addition, we recorded the longest reported expression period (400 days) of the sonoporation-delivered gene. Whole genome resequencing confirmed that the gene with stable expression existed in an extrachromosomal state without integration. Our results demonstrated the potential of sonoporation for efficient and safe DNA vaccination.

Enhanced transscleral delivery using superficial ultrasound exposure and drug-loaded hydrogel.

This study employed superficial ultrasound exposure of good ocular safety and a drug-loaded hydrogel of long residence time to enable transscleral delivery. First, we designed an acoustic adaptor to limit the ultrasound exposure depth to 1.59 mm to protect the posterior eye segments. Then, we optimized the alginate/polyacrylamide ratio (3:7) of a dual-crosslinked hydrogel to enable ultrasound-triggered release of model drug (70-kDa fluorescein isothiocyanate-conjugated dextran). Using fluorescence imaging to quantify the drug release, we showed that the developed method resulted in enhanced transscleral delivery in both ex vivo porcine scleras (2.6-fold) and in vivo rabbit scleras (2.2-fold). We also demonstrated that the method increased the drug penetration depth to the whole thickness of the sclera. In particular, the drug release efficiency increased with increasing ultrasound exposure time (1 and 3 min) and intensity (8, 19, 36, and 61 mW/cm2). Using scanning electron microscopy, we revealed that ultrasound exposure resulted in rougher surfaces and microscale rupture of the hydrogel. Moreover, Masson staining of scleral slices showed that the integrity of the top scleral fibers was disturbed by ultrasound exposure, and this disturbance recovered 3 days later. Our work demonstrates that the developed method holds great potential for mediating ocular drug delivery.

Dual Input-Controlled Synthetic mRNA Circuit for Bidirectional Protein Expression Regulation.

Synthetic mRNA circuits manipulate cell fate by controlling output protein expression via cell-specific input molecule detection. Most current circuits either repress or enhance output production upon input binding. Such binary input-output mechanisms restrict the fine-tuning of protein expression to control complex cellular events. Here we designed mRNA circuits using enhancer/repressor modules that were independently controlled by different input molecules, resulting in bidirectional output regulation; the maximal enhancement over maximal repression was 57 fold. The circuit either enhances or represses protein production in different cells based on the difference in the expression of two microRNAs. This study examined novel bidirectional circuit designs capable of fine-tuning protein production by sensing multiple input molecules. It also broadened the scope of cell manipulation by synthetic mRNA circuits, facilitating the development of mRNA circuits for precise cell manipulation and providing cell-based solutions to biomedical problems.

Effects of increased ozone on rice panicle morphology.

Ground-level ozone threatens rice production, which provides staple food for more than half of the world's population. Improving the adaptability of rice crops to ozone pollution is essential to ending global hunger. Rice panicles not only affect grain yield and grain quality but also the adaptability of plants to environmental changes, but the effects of ozone on rice panicles are not well understood. Through an open top chamber experiment, we investigated the effects of long-term and short-term ozone on the traits of rice panicles, finding that both long-term and short-term ozone significantly reduced the number of panicle branches and spikelets in rice, and especially the fertility of spikelets in hybrid cultivar. The reduction in spikelet quantity and fertility because of ozone exposure is caused by changes in secondary branches and attached spikelet. These results suggest the potential for effective adaptation to ozone by altering breeding targets and developing growth stage-specific agricultural techniques.

Barrier-breaking effects of ultrasonic cavitation for drug delivery and biomarker release.

Recently, emerging evidence has demonstrated that cavitation actually creates important bidirectional channels on biological barriers for both intratumoral drug delivery and extratumoral biomarker release. To promote the barrier-breaking effects of cavitation for both therapy and diagnosis, we first reviewed recent technical advances of ultrasound and its contrast agents (microbubbles, nanodroplets, and gas-stabilizing nanoparticles) and then reported the newly-revealed cavitation physical details. In particular, we summarized five types of cellular responses of cavitation in breaking the plasma membrane (membrane retraction, sonoporation, endocytosis/exocytosis, blebbing and apoptosis) and compared the vascular cavitation effects of three different types of ultrasound contrast agents in breaking the blood-tumor barrier and tumor microenvironment. Moreover, we highlighted the current achievements of the barrier-breaking effects of cavitation in mediating drug delivery and biomarker release. We emphasized that the precise induction of a specific cavitation effect for barrier-breaking was still challenged by the complex combination of multiple acoustic and non-acoustic cavitation parameters. Therefore, we provided the cutting-edge in-situ cavitation imaging and feedback control methods and suggested the development of an international cavitation quantification standard for the clinical guidance of cavitation-mediated barrier-breaking effects.

Identification of thioredoxin-1 as a biomarker of lung cancer and evaluation of its prognostic value based on bioinformatics analysis.

Background: Thioredoxin-1 (TXN), a redox balance factor, plays an essential role in oxidative stress and has been shown to act as a potential contributor to various cancers. This study evaluated the role of TXN in lung cancer by bioinformatics analyses. Materials and methods: Genes differentially expressed in lung cancer and oxidative stress related genes were obtained from The Cancer Genome Atlas, Gene Expression Omnibus and GeneCards databases. Following identification of TXN as an optimal differentially expressed gene by bioinformatics, the prognostic value of TXN in lung cancer was evaluated by univariate/multivariate Cox regression and Kaplan-Meier survival analyses, with validation by receiver operation characteristic curve analysis. The association between TXN expression and lung cancer was verified by immunohistochemical analysis of the Human Protein Atlas database, as well as by western blotting and qPCR. Cell proliferation was determined by cell counting kit-8 after changing TXN expression using lentiviral transfection. Results: Twenty differentially expressed oxidative stress genes were identified. Differential expression analysis identified five genes (CASP3, CAT, TXN, GSR, and HSPA4) and Kaplan-Meier survival analysis identified four genes (IL-6, CYCS, TXN, and BCL2) that differed significantly in lung cancer and normal lung tissue, indicating that TXN was an optimal differentially expressed gene. Multivariate Cox regression analysis showed that T stage (T3/T4), N stage (N2/N3), curative effect (progressive diseases) and high TXN expression were associated with poor survival, although high TXN expression was poorly predictive of overall survival. TXN was highly expressed in lung cancer tissues and cells. Knockdown of TXN suppressed cell proliferation, while overexpression of TXN enhanced cell proliferation. Conclusion: High expression of TXN plays an important role in lung cancer development and prognosis. Because it is a prospective prognostic factor, targeting TXN may have clinical benefits in the treatment of lung cancer.

GOLPH3 Promotes Vasculogenic Mimicry via the Epithelial Mesenchymal Transition in Glioblastoma Cells.

AIM: To evaluate the relationship between Golgi phosphoprotein 3 (GOLPH3) and vasculogenic mimicry (VM) in glioblastoma cells. MATERIAL AND METHODS: Glioma tissues from 40 glioma patients with different pathological grades were collected. GOLPH3 and VM were evaluated by immunostaining in glioma tissues. Then, the correlation between GOLPH3 and VM were analyzed clinically. Additionally, a GOLPH3-downregulation lentivirus was constructed and transfected into the human primary glioblastoma cell line, U-87 MG. Afterwards, apoptosis, migration and invasion were assessed to determine the effects of downregulation GOLPH3. Then, E-cadherin and matrix metalloproteinase 2 (MMP2) were detected for assessment of the epithelial mesenchymal transition (EMT). RESULTS: GOLPH3 and VM were found to be positively correlated following clinical analysis (p < 0.01, r=0.788). Furthermore, GOLPH3 downregulation suppressed the migration and invasion of U87 MG cells (p < 0.05), followed by upregulation of E-cadherin and downregulation of MMP2. CONCLUSION: Collectively, our results demonstrate that GOLPH3 promoted VM in glioblastoma cells and that the corresponding mechanism was associated with the EMT. Our finding suggests that GOLPH3 may represent a promising therapeutic target for mitigating the recurrence and invasion of gliomas.

Cytidine-containing tails robustly enhance and prolong protein production of synthetic mRNA in cell and in vivo.

Synthetic mRNAs are rising rapidly as alternative therapeutic agents for delivery of proteins. However, the practical use of synthetic mRNAs has been restricted by their low cellular stability as well as poor protein production efficiency. The key roles of poly(A) tail on mRNA biology inspire us to explore the optimization of tail sequence to overcome the aforementioned limitations. Here, the systematic substitution of non-A nucleotides in the tails revealed that cytidine-containing tails can substantially enhance the protein production rate and duration of synthetic mRNAs both in vitro and in vivo. Such C-containing tails shield synthetic mRNAs from deadenylase CCR4-NOT transcription complex, as the catalytic CNOT proteins, especially CNOT6L and CNOT7, have lower efficiency in trimming of cytidine. Consistently, these enhancement effects of C-containing tails were observed on all synthetic mRNAs tested and were independent of transfection reagents and cell types. As the C-containing tails can be used along with other mRNA enhancement technologies to synergically boost protein production, we believe that these tails can be broadly used on synthetic mRNAs to directly promote their clinical applications.

Development of field-applicable endogenous internally controlled recombinase-aided amplification (EIC-RAA) assays for the detection of human papillomavirus genotypes 6 and 11 using sample releasing agent.

Objective: Human papillomavirus (HPV) 6 and 11 are the two most common low-risk HPV subtypes, accounting for more than 90% of condyloma acuminatum. A simple, accurate and rapid screening method to be applied in community-level hospitals is in high demand. Methods: Endogenous internally controlled recombinase-assisted amplification (EIC-RAA) assays for HPV6 and 11 were performed in a single closed-tube at 39 °C within 30 min. The sensitivity and specificity of EIC-RAA were examined using recombinant plasmids and pre-tested HPV DNA. A total of 233 clinical samples were collected, and the DNA was extracted by traditional multi-step extraction, or sample releasing agent, before analysis by EIC-RAA. For comparison, HPV detection via Quantitative real-time PCR (qPCR) was also performed. Results: The sensitivity of EIC-RAA analysis was 10 copies/reaction for HPV6, 100 copies/reaction for HPV11, and 100 copies/reaction for the human ß-globin gene. No cross-reaction was observed with other HPV subtypes. Clinical performance of the EIC-RAA assay achieved a 100% of concordance rate with the commercial HPV qPCR kit. Further, the EIC-RAA assay achieved a 100% of concordance rate when using multi-step extracted DNA and sample releasing agent-processed DNA. Summary: The EIC-RAA assay for HPV6 and 11 detection possesses the advantages of accuracy, simplicity and rapidity, and demonstrates great potential to be used in community-level hospitals for field investigation.

Food Security in China: A Brief View of Rice Production in Recent 20 Years.

Rice production affects the food security and socioeconomic status of over half the world's population. Rice-producing countries, however, are facing population growth, reduction in rice planting area, and global change. Understanding the trends of rice production and major determinants is key to regulating rice production. We thus analyzed the trends of rice production and related determinants in China from 2001 to 2021, revealing that the annual rice production (TRP) has risen steadily (r = 0.929, p < 0.0001) in recent 20 years. TRP in 2021 was 19.9% higher than that in 2001, which was primarily achieved by the increment of middle rice production (MRP). MRP increased by 46.2% from 2000 to 2018, and grain yield per unit area (GPA) was the largest in middle rice. The enhancement of GPAs is significantly correlated with the consumption of agricultural resources and the number of released rice cultivars, but variations exist. TRP and GPA vary in different provinces; Hunan (25 ± 2 megatons) and Xinjiang (8364 ± 806 kg/hectare) show the largest values, respectively. TRP could be further increased by 13.8% by improving MRP. The results suggest that rice production in China has a large potential to be further improved through regulations.

Ethylenediurea reduces grain chalkiness in hybrid rice cultivars under ambient levels of surface ozone in China.

High concentration of tropospheric ozone (O3) causes crop yield losses, which could be reduced by foliar application of ethylenediurea (EDU). Rice grain appearance is a major quality trait that determines the milling quality, white rice productivity and the market value. Grain chalkiness is one of the common defects that deteriorate the grain appearance in rice due to its negative effects on palatability and milling yield. Whether EDU could reduce grain chalkiness in rice which was usually increased by high concentration of O3 is not clarified. We report the grain chalkiness in 19 rice cultivars (CVs) of three types: indica (6 CVs), japonica (5 CVs) and hybrids (8 CVs), observed in an EDU application experiment in the field in China. The ambient O3 level as expressed by accumulated hourly O3 concentration over the threshold of 40 ppb (AOT40) for 80 days until maturity reached 12.8 ppm h at a near-by monitoring station. Fraction of the chalky grains (FCG) in the hybrid cultivars was 8% lower in EDU than that in the control treatments, whereas no significant effect of EDU on FCG was found in japonica or indica cultivars. The reduction of FCG due to EDU treatment in hybrid cultivars was attributed to the significant reduction of milky white grains followed by that of white belly grains. Thus, the application of EDU could ameliorate the decline of grain appearance quality in hybrid rice by decreasing the FCG and enhancing the fraction of perfect grains (FPG). Moreover, there were significant interactions between the EDU application and rice cultivars, indicating varietal difference in the protection of grain appearance quality by EDU. These results suggest the need for further studies on the mechanisms of the effects of EDU on grain chalkiness.

Fabrication of lignin reinforced hybrid hydrogels with antimicrobial and self-adhesion for strain sensors.

Ionic conductive hydrogels prepared from various biological macromolecules are ideal materials for the manufacture of human motion sensors from the perspective of resource regeneration and environmental sustainability. However, it is still challenging to prepare hydrogels with both high toughness and self-healing ability. In this study, lignin-based ß-CD-PVA (LCP) self-healing conductive hydrogels with high tensile properties were prepared by one-step method using alkali lignin as a plasticizer. Compared with PVA hydrogel, the maximum storage modulus and elongation were increased by 2.5 and 20.0 times, respectively. Uniform distribution of lignin can increase the fluidity and distance of polymer molecular chains, thus improving the viscoelastic and tensile properties of the LCP self-healing hydrogel. LCP hydrogels can maintain self-healing ability in both high (45 °C) and low temperature (0 °C) environments, and the self-healing ability is not affected by pH. Moreover, it also has good conductivity, anti-bacterial, thermostability, and anti-UV property, which has a good application prospect in the field of 3D printing and wearable electronic devices, which expands the efficient utilization of lignin in biorefinery.

Efficacy and Safety of Moxibustion for Menopausal Obesity: A Multicentre, Randomized, Controlled Trial Protocol.

Background. In the past, moxibustion has been widely used to treat endocrine system disorders, but evidence of its effectiveness is scarce at this point. The aim of this multicenter, randomized, controlled trial is to evaluate the efficacy and safety of treating menopausal obesity with moxibustion. Methods/Design. There are six centers taking part in this randomized, controlled, parallel trial. A total of 216 patients with menopausal obesity will be randomly divided into two equal groups: the "moxibustion for harmonization of Yin and Yang" group and the gentle moxibustion group. A 12-week study period with moxibustion will be preceded by a 1-week baseline, followed by a 12-week follow-up. We will conduct an interim analysis to determine whether or not the treatment is efficacious and safe after 216 participants have completed a 12-week treatment period. Evaluations will be conducted at weeks 0, 4, 8, 12, 18, and 24. The main outcome is waist circumference (WC), and the rate of WC reduction will be compared to the baseline. An intention-to-treat analysis will be performed with a two-sided P value of <0.05 considered significant. Participants who withdraw from the trial will be analyzed according to the intention-to-treat formula (ITT). Discussion. These results will be used to support selecting the right moxibustion prescription and guiding the improvement of clinical efficacy. This trial will provide convincing evidence of moxibustion's effectiveness and safety in the treatment of obesity by "moxibustion for harmonization of Yin and Yang," which will be conducive to the promotion and clinical application of the theory of "moxibustion for harmonization of Yin and Yang." Trial Registration. This trial is registered with Clinical Trials.gov: NCT04943705 (registered on June 27, 2021).

DCBLD2 regulates vascular hyperplasia by modulating the platelet derived growth factor receptor-ß endocytosis through Caveolin-1 in vascular smooth muscle cells.

DCBLD2 is a neuropilin-like transmembrane protein that is up-regulated during arterial remodeling in humans, rats, and mice. Activation of PDGFR-ß via PDGF triggers receptor phosphorylation and endocytosis. Subsequent activation of downstream signals leads to the stimulation of phenotypic conversion of VSMCs and arterial wall proliferation, which are common pathological changes in vascular remodeling diseases such as atherosclerosis, hypertension, and restenosis after angioplasty. In this study, we hypothesized that DCBLD2 regulates neointimal hyperplasia through the regulation of PDGFR-ß endocytosis of vascular smooth muscle cells (VSMCs) through Caveolin-1 (Cav-1). Compared with wild-type (WT) mice or control littermate mice, the germline or VSMC conditional deletion of the Dcbld2 gene resulted in a significant increase in the thickness of the tunica media in the carotid artery ligation. To elucidate the underlying molecular mechanisms, VSMCs were isolated from the aorta of WT or Dcbld2-/- mice and were stimulated with PDGF. Western blotting assays demonstrated that Dcbld2 deletion increased the PDGF signaling pathway. Biotin labeling test and membrane-cytosol separation test showed that after DCBLD2 was knocked down or knocked out, the level of PDGFR-ß on the cell membrane was significantly reduced, while the amount of PDGFR-ß in the cytoplasm increased. Co-immunoprecipitation experiments showed that after DCBLD2 gene knock-out, the binding of PDGFR-ß and Cav-1 in the cytoplasm significantly increased. Double immunofluorescence staining showed that PDGFR-ß accumulated Cav-1/lysosomes earlier than for control cells, which indicated that DCBLD2 gene knock-down or deletion accelerated the endocytosis of PDGF-induced PDGFR-ß in VSMCs. In order to confirm that DCBLD2 affects the relationship between Cav-1 and PDGFR-ß, proteins extracted from VSMCs cultured in vitro were derived from WT and Dcbld2-/- mice, whereas co-immunoprecipitation suggested that the combination of DCBLD2 and Cav-1 reduced the bond between Cav-1 and PDGFR-ß, and DCBLD2 knock-out was able to enhance the interaction between Cav-1 and PDGFR-ß. Therefore, the current results suggest that DCBLD2 may inhibit the caveolae-dependent endocytosis of PDGFR-ß by anchoring the receptor on the cell membrane. Based on its ability to regulate the activity of PDGFR-ß, DCBLD2 may be a novel therapeutic target for the treatment of cardiovascular diseases.

Transparent, photothermal and stretchable alginate-based hydrogels for remote actuation and human motion sensing.

Multifunctional hydrogels show potential applications in actuators and wearable sensors. However, it is still a challenge to develop a photothermal responsive conductive hydrogel with high transparency, mechanical properties, broad sensing range, and low-temperature resistance. In this work, a transparent, photothermal responsive, and highly stretchable alginate-based hydrogels was feasibly constructed by adding two-dimensional non-layered molybdenum dioxide nanosheets (2D-MoO2) to sodium alginate/polyacrylamide mixture and then soaking into the calcium chloride solution. The introduction of 2D-MoO2 renders the hydrogels excellent photothermal properties and controllable photomechanical deformation under near-infrared irradiation, while maintaining high transparency (~60 %).The calcium ions give the hydrogel excellent mechanics, conductivity, and freezing tolerance concurrently. The transparent hydrogel-based sensor shows wide sensing range (0-1800 %) and cycling stability in detecting deformations and real-time human motions even in harsh environments. Therefore, this work provides a new route for generating transparent multifunctional hydrogels towards the applications of remote actuation and strain sensing.

Development of a duplex recombinase-aided amplification assay for direct detection of Mycoplasma pneumoniae and Chlamydia trachomatis in clinical samples.

BACKGROUND: Pneumonia caused by Mycoplasma pneumoniae is common in the elderly and children, and pneumonia caused by Chlamydia trachomatis is prevalent in newborns. This study aimed to establish a rapid, sensitive, and simple method for the direct detection of M. pneumoniae and C. trachomatis in clinical samples without DNA extraction. METHODS: We established a duplex recombinase-aided amplification (RAA) assay with the RNAseP gene as an internal control for detecting the P1 gene of M. pneumoniae and the ORF8 gene of C. trachomatis, respectively. The results were obtained at 39 °C within 15-20 min. A total of 130 clinical samples suspected of M. pneumoniae or C. trachomatis infection were collected and tested by duplex RAA and PCR. DNA extracted via a commercial kit or treated with a nucleic acid-releasing agent was used and compared, respectively. Standard recombinant plasmids were used to test the sensitivity of the duplex RAA assay. In addition, other similar common pathogens were used to verify the specificity of the duplex RAA assay. RESULTS: The sensitivity of the duplex RAA assay for detecting M. pneumoniae and C. trachomatis was 10 copies/µL using recombinant plasmids. Compared with PCR, the sensitivity and specificity of duplex RAA assays for M. pneumoniae and C. trachomatis was 100% using clinical DNA samples extracted using a commercial kit and a nucleic acid-releasing agent, and the Kappa value was 1. CONCLUSION: The advantages of this duplex RAA assay include high sensitivity and specificity, short duration, and simple extraction steps, with potential for use in the on-site detection of M. pneumoniae and C. trachomatis in resource-limited settings.

Aptamer-Array-Guided Protein Assembly Enhances Synthetic mRNA Switch Performance.

Synthetic messenger RNA (mRNA) switches are powerful synthetic biological tools that can sense cellular molecules to manipulate cell fate. However, their performances are limited by high output signal noise due to leaky output protein expression. Here, we designed a readout control module that disables protein leakage from generating signal. Aptamer array on the switch guides the inactive output protein to self-assemble into functional assemblies that generate output signal. Leaky protein expression fails to saturate the array, thus produces marginal signal. In this study, we demonstrated that switches with this module exhibit substantially lower signal noise and, consequently, higher input sensitivity and wider output range. Such switches are applicable for different types of input molecules and output proteins. The work here demonstrates a new type of spatially guided protein self-assembly, affording novel synthetic mRNA switches that promise accurate cell manipulation for biomedical applications.

Using Smart Displays to Implement an eHealth System for Older Adults With Multiple Chronic Conditions: Protocol for a Randomized Controlled Trial.

BACKGROUND: Voice-controlled smart speakers and displays have a unique but unproven potential for delivering eHealth interventions. Many laptop- and smartphone-based interventions have been shown to improve multiple outcomes, but voice-controlled platforms have not been tested in large-scale rigorous trials. Older adults with multiple chronic health conditions, who need tools to help with their daily management, may be especially good candidates for interventions on voice-controlled devices because these patients often have physical limitations, such as tremors or vision problems, that make the use of laptops and smartphones challenging. OBJECTIVE: The aim of this study is to assess whether participants using an evidence-based intervention (ElderTree) on a smart display will experience decreased pain interference and improved quality of life and related measures in comparison with participants using ElderTree on a laptop and control participants who are given no device or access to ElderTree. METHODS: A total of 291 adults aged ≥60 years with chronic pain and ≥3 additional chronic conditions will be recruited from primary care clinics and community organizations and randomized 1:1:1 to ElderTree access on a smart display along with their usual care, ElderTree access on a touch screen laptop along with usual care, or usual care alone. All patients will be followed for 8 months. The primary outcomes are differences between groups in measures of pain interference and psychosocial quality of life. The secondary outcomes are between-group differences in system use at 8 months, physical quality of life, pain intensity, hospital readmissions, communication with medical providers, health distress, well-being, loneliness, and irritability. We will also examine mediators and moderators of the effects of ElderTree on both platforms. At baseline, 4 months, and 8 months, patients will complete written surveys comprising validated scales selected for good psychometric properties with similar populations. ElderTree use data will be collected continuously in system logs. We will use linear mixed-effects models to evaluate outcomes over time, with treatment condition and time acting as between-participant factors. Separate analyses will be conducted for each outcome. RESULTS: Recruitment began in August 2021 and will run through April 2023. The intervention period will end in December 2023. The findings will be disseminated via peer-reviewed publications. CONCLUSIONS: To our knowledge, this is the first study with a large sample and long time frame to examine whether a voice-controlled smart device can perform as well as or better than a laptop in implementing a health intervention for older patients with multiple chronic health conditions. As patients with multiple conditions are such a large cohort, the implications for cost as well as patient well-being are significant. Making the best use of current and developing technologies is a critical part of this effort. TRIAL REGISTRATION: ClinicalTrials.gov NCT04798196; https://clinicaltrials.gov/ct2/show/NCT04798196. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/37522.