Elite rhetoric can undermine democratic norms.

Democratic stability depends on citizens on the losing side accepting election outcomes. Can rhetoric by political leaders undermine this norm? Using a panel survey experiment, we evaluate the effects of exposure to multiple statements from former president Donald Trump attacking the legitimacy of the 2020 US presidential election. Although exposure to these statements does not measurably affect general support for political violence or belief in democracy, it erodes trust and confidence in elections and increases belief that the election is rigged among people who approve of Trump's job performance. These results suggest that rhetoric from political elites can undermine respect for critical democratic norms among their supporters.

Immobilization of azide-functionalized proteins to micro- and nanoparticles directly from cell lysate.

Immobilization of proteins and enzymes on solid supports has been utilized in a variety of applications, from improved protein stability on supported catalysts in industrial processes to fabrication of biosensors, biochips, and microdevices. A critical requirement for these applications is facile yet stable covalent conjugation between the immobilized and fully active protein and the solid support to produce stable, highly bio-active conjugates. Here, we report functionalization of solid surfaces (gold nanoparticles and magnetic beads) with bio-active proteins using site-specific and biorthogonal labeling and azide-alkyne cycloaddition, a click chemistry. Specifically, we recombinantly express and selectively label calcium-dependent proteins, calmodulin and calcineurin, and cAMP-dependent protein kinase A (PKA) with N-terminal azide-tags for efficient conjugation to nanoparticles and magnetic beads. We successfully immobilized the proteins on to the solid supports directly from the cell lysate with click chemistry, forgoing the step of purification. This approach is optimized to yield low particle aggregation and high levels of protein activity post-conjugation. The entire process enables streamlined workflows for bioconjugation and highly active conjugated proteins.

Towards the use of a smartphone imaging-based tool for point-of-care detection of asymptomatic low-density malaria parasitaemia.

BACKGROUND: Globally, there are over 200 million cases of malaria annually and over 400,000 deaths. Early and accurate detection of low-density parasitaemia and asymptomatic individuals is key to achieving the World Health Organization (WHO) 2030 sustainable development goals of reducing malaria-related deaths by 90% and eradication in 35 countries. Current rapid diagnostic tests are neither sensitive nor specific enough to detect the low parasite concentrations in the blood of asymptomatic individuals. METHODS: Here, an imaging-based sensing technique, particle diffusometry (PD), is combined with loop mediated isothermal amplification (LAMP) on a smartphone-enabled device to detect low levels of parasitaemia often associated with asymptomatic malaria. After amplification, PD quantifies the Brownian motion of fluorescent nanoparticles in the solution during a 30 s video taken on the phone. The resulting diffusion coefficient is used to detect the presence of Plasmodium DNA amplicons. The coefficients of known negative samples are compared to positive samples using a one-way ANOVA post-hoc Dunnett's test for confirmation of amplification. RESULTS: As few as 3 parasite/µL of blood was detectable in 45 min without DNA extraction. Plasmodium falciparum parasites were detected from asymptomatic individuals' whole blood samples with 89% sensitivity and 100% specificity when compared to quantitative polymerase chain reaction (qPCR). CONCLUSIONS: PD-LAMP is of value for the detection of low density parasitaemia especially in areas where trained personnel may be scarce. The demonstration of this smartphone biosensor paired with the sensitivity of LAMP provides a proof of concept to achieve widespread asymptomatic malaria testing at the point of care.

Usability of Rapid Cholera Detection Device (OmniVis) for Water Quality Workers in Bangladesh: Iterative Convergent Mixed Methods Study.

BACKGROUND: Cholera poses a significant global health burden. In Bangladesh, cholera is endemic and causes more than 100,000 cases each year. Established environmental reservoirs leave millions at risk of infection through the consumption of contaminated water. The Global Task Force for Cholera Control has called for increased environmental surveillance to detect contaminated water sources prior to human infection in an effort to reduce cases and deaths. The OmniVis rapid cholera detection device uses loop-mediated isothermal amplification and particle diffusometry detection methods integrated into a handheld hardware device that attaches to an iPhone 6 to identify and map contaminated water sources. OBJECTIVE: The aim of this study was to evaluate the usability of the OmniVis device with targeted end users to advance the iterative prototyping process and ultimately design a device that easily integrates into users' workflow. METHODS: Water quality workers were trained to use the device and subsequently completed an independent device trial and usability questionnaire. Pretraining and posttraining knowledge assessments were administered to ensure training quality did not confound trial and questionnaire. RESULTS: Device trials identified common user errors and device malfunctions including incorrect test kit insertion and device powering issues. We did not observe meaningful differences in user errors or device malfunctions accumulated per participant across demographic groups. Over 25 trials, the mean time to complete a test was 47 minutes, a significant reduction compared with laboratory protocols, which take approximately 3 days. Overall, participants found the device easy to use and expressed confidence and comfort in using the device independently. CONCLUSIONS: These results are used to advance the iterative prototyping process of the OmniVis rapid cholera detection device so it can achieve user uptake, workflow integration, and scale to ultimately impact cholera control and elimination strategies. We hope this methodology will promote robust usability evaluations of rapid pathogen detection technologies in device development.

DNA Microviscosity Characterization with Particle Diffusometry for Downstream DNA Detection Applications.

Analytical characterization of DNA microviscosity provides critical biophysical insights into nuclear crowding, nucleic acid based pharmaceutical development, and nucleic acid based biosensor device design. However, most viscosity characterization methods require large sample volumes and destructive testing. In contrast, particle diffusometry permits in situ analysis of DNA microviscosity with short measurement times (8 s) using small volumes (<3 µL) which are compatible with DNA preparatory procedures. This unconventional biosensing approach involves measuring the change in sample viscosity using image processing and correlation-based algorithms. Particle diffusometry requires only a fluorescence microscope equipped with a charge-coupled device (CCD) camera and is a nondestructive measurement method. We use particle diffusometry to characterize the effect of DNA topology, length, and concentration on solution viscosity. In addition, we use particle diffusometry to detect the amplification of DNA from Staphylococcus aureus and Klebsiella pneumoniae, two pathogens commonly related to neonatal sepsis. Successful characterization of pathogen amplification with particle diffusometry provides a new opportunity to apply viscosity characterization toward downstream applications in nucleic acid based pathogen detection.

Optoelectric patterning: Effect of electrode material and thickness on laser-induced AC electrothermal flow.

Rapid electrokinetic patterning (REP) is an emerging optoelectric technique that takes advantage of laser-induced AC electrothermal flow and particle-electrode interactions to trap and translate particles. The electrothermal flow in REP is driven by the temperature rise induced by the laser absorption in the thin electrode layer. In previous REP applications 350-700 nm indium tin oxide (ITO) layers have been used as electrodes. In this study, we show that ITO is an inefficient electrode choice as more than 92% of the irradiated laser on the ITO electrodes is transmitted without absorption. Using theoretical, computational, and experimental approaches, we demonstrate that for a given laser power the temperature rise is controlled by both the electrode material and its thickness. A 25-nm thick Ti electrode creates an electrothermal flow of the same speed as a 700-nm thick ITO electrode while requiring only 14% of the laser power used by ITO. These results represent an important step in the design of low-cost portable REP systems by lowering the material cost and power consumption of the system.

Real-Time Visualization of HIV-1 RNA Detection Using Loop-Mediated Isothermal Amplification-Enabled Particle Diffusometry.

Isothermal nucleic acid amplification tests, NAATs, such as reverse transcription-loop-mediated isothermal amplification (RT-LAMP), offer promising capabilities to perform real-time semiquantitative detection of viral pathogens. These tests provide rapid results, utilize simple instrumentation for single-temperature reactions, support efficient user workflows, and are suitable for field use. Herein, we present a novel and robust method for real-time monitoring of HIV-1 RNA RT-LAMP utilizing a novel implementation of particle diffusometry (PD), a diffusivity quantification technique using fluorescent particles, to quantify viral concentration in nuclease-free water. We monitor changes in particle diffusion dynamics of 400 nm fluorescently labeled particles throughout the RT-LAMP of HIV-1 RNA in nuclease-free water, enabling measurement within 20 min and detection of concentrations as low as 25 virus particles per µL. Moreover, in a single-blind study, we demonstrate semiquantitative detection by accurately determining the initial concentration of an unknown HIV-1 RNA within a 10% absolute error margin. These results highlight the potential of real-time PD readout for quantifying HIV-1 RNA via RT-LAMP, offering promise for viral load monitoring of HIV and other chronic infections.

A remarkable short-snouted horned dinosaur from the Late Cretaceous (late Campanian) of southern Laramidia.

The fossil record of centrosaurine ceratopsids is largely restricted to the northern region of western North America (Alberta, Montana and Alaska). Exceptions consist of single taxa from Utah (Diabloceratops) and China (Sinoceratops), plus otherwise fragmentary remains from the southern Western Interior of North America. Here, we describe a remarkable new taxon, Nasutoceratops titusi n. gen. et sp., from the late Campanian Kaiparowits Formation of Utah, represented by multiple specimens, including a nearly complete skull and partial postcranial skeleton. Autapomorphies include an enlarged narial region, pneumatic nasal ornamentation, abbreviated snout and elongate, rostrolaterally directed supraorbital horncores. The subrectangular parietosquamosal frill is relatively unadorned and broadest in the mid-region. A phylogenetic analysis indicates that Nasutoceratops is the sister taxon to Avaceratops, and that a previously unknown subclade of centrosaurines branched off early in the group's history and persisted for several million years during the late Campanian. As the first well-represented southern centrosaurine comparable in age to the bulk of northern forms, Nasutoceratops provides strong support for the provincialism hypothesis, which posits that Laramidia-the western landmass formed by inundation of the central region of North America by the Western Interior Seaway-hosted at least two coeval dinosaur communities for over a million years of late Campanian time.

Quantifying Brownian motion in the presence of simple shear flow with particle diffusometry.

Particle diffusometry, a technology derived from particle image velocimetry, quantifies the Brownian motion of particles suspended in a quiescent solution by computing the diffusion coefficient. Particle diffusometry has been used for pathogen detection by measuring the change in solution viscosity due to amplified DNA from a specific gene target. However, particle diffusometry fails to calculate accurate measurements at elevated temperatures and fluid flow. Therefore, these two current limitations hinder the potential application where particle diffusometry can further be used. In this work, we expanded the usability of particle diffusometry to be applied to fluid samples with simple shear flow and at various temperatures. A range of diffusion coefficient videos is created to simulate the Brownian motion of particles under flow and temperature conditions. Our updated particle diffusometry analysis forms a correction equation under three different polynomial degrees of shear flow with varying flow rates and temperatures between 25 and 65 °C. An experiment in a channel with a rectangular cross section using a syringe pump to generate a constant flow is done to analyze the modified algorithm. In simulation analysis, the modified algorithm successfully computes the diffusion coefficients with ±  10% error for an average flow rate of up to 8 pixel / Δ t on all three flow types. Complementary experiments confirm the simulation results.

PD-LAMP smartphone detection of SARS-CoV-2 on chip.

In 2019 the COVID-19 pandemic, caused by SARS-CoV-2, demonstrated the urgent need for rapid, reliable, and portable diagnostics. The COVID-19 pandemic was declared in January 2020 and surges of the outbreak continue to reoccur. It is clear that early identification of infected individuals, especially asymptomatic carriers, plays a huge role in preventing the spread of the disease. The current gold standard diagnostic for SARS-CoV-2 is quantitative reverse transcription polymerase chain reaction (qRT-PCR) test based on the detection of the viral RNA. While RT-PCR is reliable and sensitive, it requires expensive centralized equipment and is time consuming (∼2 h or more); limiting its applicability in low resource areas. The FDA issued Emergency Use Authorizations (EUAs) for several COVID-19 diagnostics with an emphasis on point-of care (PoC) testing. Numerous RT-PCR and serological tests were approved for use at the point of care. Abbott's ID NOW, and Cue Health's COVID-19 test are of particular interest, which use isothermal amplification methods for rapid detection in under 20 min. We look to expand on the range of current PoC testing platforms with a new rapid and portable isothermal nucleic acid detection device. We pair reverse transcription loop mediated isothermal amplification (RT-LAMP) with a particle imaging technique, particle diffusometry (PD), to successfully detect SARS-CoV-2 in only 35 min on a portable chip with integrated heating. A smartphone device is used to image the samples containing fluorescent beads post-RT-LAMP and correlates decreased diffusivity to positive samples. We detect as little as 30 virus particles per µL from a RT-LAMP reaction in a microfluidic chip using a portable heating unit. Further, we can perform RT-LAMP from a diluted unprocessed saliva sample without RNA extraction. Additionally, we lyophilize SARS-CoV-2-specific RT-LAMP reactions that target both the N gene and the ORF1ab gene in the microfluidic chip, eliminating the need for cold storage. Our assay meets specific target product profiles outlined by the World Health Organization: it is specific to SARS-CoV-2, does not require cold storage, is compatible with digital connectivity, and has a detection limit of less than 35 × 104 viral particles per mL in saliva. PD-LAMP is rapid, simple, and attractive for screening and use at the point of care.

Evaluating the effects of vaccine messaging on immunization intentions and behavior: Evidence from two randomized controlled trials in Vermont.

The effectiveness of vaccines in reducing child morbidity and mortality worldwide relies on public acceptance. However, relatively little is known about the effects of vaccine communication on vaccine attitudes and immunization behavior. Previous research suggests that common communication approaches may be ineffective or even counterproductive, especially among vaccine-hesitant parents. However, these studies typically rely on observational data or self-reported measures of vaccination intention. Using novel research designs, we tested the attitudinal and behavioral effects of messages encouraging vaccination in both a survey experiment conducted among a large sample of parents in Vermont who expressed hesitancy about childhood immunizations and a field experiment among parents whose children were overdue for vaccines. We find that neither a message promoting immunization as a social norm nor a message correcting common misperceptions about vaccines was measurably more effective than a standard public health message at improving parents' attitudes toward vaccines, intention to vaccinate their children, or compliance with the recommended vaccine schedule. Our results highlight the need for more research on approaches to successfully reducing vaccine hesitancy among parents.

A smartphone-based particle diffusometry platform for sub-attomolar detection of Vibrio cholerae in environmental water.

Each year, 3.4 million people die from waterborne diseases worldwide. Development of a rapid and portable platform for detecting and monitoring waterborne pathogens would significantly aid in reducing the incidence and spread of infectious diseases. By combining optical methods and smartphone technology with molecular assays, the sensitivity required to detect exceedingly low concentrations of waterborne pathogens can readily be achieved. Here, we implement smartphone-based particle diffusometry (PD) detection of loop-mediated isothermal amplification (LAMP) targeting the waterborne pathogen Vibrio cholerae (V. cholerae). By measuring the diffusion of 400 nm streptavidin-coated fluorescent nanoparticles imaged at 68X magnification on a smartphone, we can detect as few as 6 V. cholerae cells per reaction (0.66 aM) in just 35 minutes. In a double-blinded study with 132 pond water samples, we establish a 91.8% sensitivity, 95.2% specificity, and 94.3% accuracy of the smartphone-based PD platform for detection of V. cholerae. Together, these results demonstrate the utility of this smartphone-based PD platform for rapid and sensitive detection of V. cholerae at the point of use.

Particle Diffusometry: An Optical Detection Method for Vibrio cholerae Presence in Environmental Water Samples.

There is a need for a rapid, robust, and sensitive biosensor to identify low concentrations of pathogens in their native sample matrix without enrichment or purification. Nucleic acid-based detection methods are widely accepted as the gold standard in diagnostics, but robust detection of low concentrations of pathogens remains challenging. Amplified nucleic acids produce more viscous solutions, which can be measured by combining these products with fluorescent particles and measuring the change in the particle diffusion coefficient using a technique known as particle diffusometry. Here, we utilize Vibrio cholerae (V. cholerae) as a proof-of-concept for our detection system due to its inherently low concentration in environmental water samples. We demonstrate that particle diffusometry can be used to detect down to 1 V. cholerae cell in molecular-grade water in 20 minutes and 10 V. cholerae cells in pond water in just 35 minutes in 25 µL reaction volumes. The detection limit in pond water is environmentally relevant and does not require any enrichment or sample preparation steps. Particle diffusometry is 10-fold more sensitive than current gold standard fluorescence detection of nucleic acid amplification. Therefore, this novel measurement technique is a promising approach to detect low levels of pathogens in their native environments.

Dynamic optoelectric trapping and deposition of multiwalled carbon nanotubes.

In the path toward the realization of carbon nanotube (CNT)-driven electronics and sensors, the ability to precisely position CNTs at well-defined locations remains a significant roadblock. Highly complex CNT-based bottom-up structures can be synthesized if there is a method to accurately trap and place these nanotubes. In this study, we demonstrate that the rapid electrokinetic patterning (REP) technique can accomplish these tasks. By using laser-induced alternating current (AC) electrothermal flow and particle-electrode forces, REP can collect and maneuver a wide range of vertically aligned multiwalled CNTs (from a single nanotube to over 100 nanotubes) on an electrode surface. In addition, these trapped nanotubes can be electrophoretically deposited at any desired location onto the electrode surface. Apart from active control of the position of these deposited nanotubes, the number of CNTs in a REP trap can also be dynamically tuned by changing the AC frequency or by adjusting the concentration of the dispersed nanotubes. On the basis of a calculation of the stiffness of the REP trap, we found an upper limit of the manipulation speed, beyond which CNTs fall out of the REP trap. This peak manipulation speed is found to be dependent on the electrothermal flow velocity, which can be varied by changing the strength of the AC electric field.

Physical characterization of nanoparticle size and surface modification using particle scattering diffusometry.

As the field of colloidal science continues to expand, tools for rapid and accurate physiochemical characterization of colloidal particles will become increasingly important. Here, we present Particle Scattering Diffusometry (PSD), a method that utilizes dark field microscopy and the principles of particle image velocimetry to measure the diffusivity of particles undergoing Brownian motion. PSD measures the diffusion coefficient of particles as small as 30 nm in diameter and is used to characterize changes in particle size and distribution as a function of small, label-free, surface modifications of particles. We demonstrate the rapid sizing of particles using three orders-of-magnitude less sample volume than current standard techniques and use PSD to quantify particle uniformity. Furthermore, PSD is sensitive enough to detect biomolecular surface modifications of nanometer thickness. With these capabilities, PSD can reliably aid in a wide variety of applications, including colloid sizing, particle corona characterization, protein footprinting, and quantifying biomolecule activity.