Consistent Visual Analysis of Multielement Data: A Preliminary Evaluation.

Experimenters provided 33 graphical displays of hypothetical data depicted in a multielement experimental design to editorial board members of prominent, applied, behavior-analytic journals via an online survey. For each display, participants indicated (a) the presence or absence of experimental control and (b) the degree of experimental control (rated on a 1-100 scale). Each depiction varied systematically in (a) the number of data paths, (b) the number of data paths elevated above the control, (c) the mean difference between affected data paths and control conditions, and (d) the degree of variability within conditions. Correspondence among experts' ratings of experimental control was high across all presented graphical displays, supporting the reliability of visual analysis as an evaluative tool for these designs.

Comparing Multiple Methods to Measure Procedural Fidelity of Discrete-trial Instruction.

Procedural fidelity is the extent to which an intervention is implemented as designed and is an important component of research and practice. There are multiple ways to measure procedural fidelity, and few studies have explored how procedural fidelity varies based on the method of measurement. The current study compared adherence to discrete-trial instruction protocols by behavior technicians with a child with autism when observers used different procedural-fidelity measures. We collected individual-component and individual-trial fidelity with an occurrence-nonoccurrence data sheet and compared these scores to global fidelity and all-or-nothing, 3-point Likert scale, and 5-point Likert scale measurement methods. The all-or-nothing method required all instances of a component or trial be implemented without error to be scored correct. The Likert scales used a rating system to score components and trials. At the component level, we found that the global, 3-point Likert, and 5-point Likert methods were likely to overestimate fidelity and mask component errors, and the all-or-nothing method was unlikely to mask errors. At the trial level, we found that the global and 5-point Likert methods approximated individual-trial fidelity, the 3-point Likert method overestimated fidelity, and the all-or-nothing method underestimated fidelity. The occurrence-nonoccurrence method required the most time to complete, and all-or-nothing by trial required the least. We discuss the implications of measuring procedural fidelity with different methods of measurement, including false positives and false negatives, and provide suggestions for practice and research. Supplementary Information: The online version contains supplementary material available at 10.1007/s43494-023-00094-w.

The Effects of Competing Verbal Behavior on Performance in a Math Task.

The purposes of this study were to evaluate the effects of an auditory-distractor stimulus and vocal-blocking task on performance on a math task and measures of overt verbal operants. College students served as participants, and they were instructed to solve an arithmetic problem while continuously emitting overt verbal behavior. The overt verbal behavior consisted of either talking aloud while solving the problem or reciting the alphabet. A third condition consisted of playing an auditory-distractor file containing the alphabet during the response interval while participants talked aloud. Data were collected on response accuracy, latency to respond, and frequency of echoic and self-echoic responses emitted during the response interval. The vocal-blocking task significantly affected performance on the math task when visual stimuli were absent. The decrease in performance coincided with significant decreases in participants' mean frequency of echoic and self-echoic responses. In sum, correctly responding to an arithmetic problem was significantly affected by the vocal-blocking task, indicating support for the necessity of verbal mediation during problem solving.

Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning.

While the immediate and transitory response of breast cancer cells to pathological stiffness in their native microenvironment has been well explored, it remains unclear how stiffness-induced phenotypes are maintained over time after cancer cell dissemination in vivo. Here, we show that fibrotic-like matrix stiffness promotes distinct metastatic phenotypes in cancer cells, which are preserved after transition to softer microenvironments, such as bone marrow. Using differential gene expression analysis of stiffness-responsive breast cancer cells, we establish a multigenic score of mechanical conditioning (MeCo) and find that it is associated with bone metastasis in patients with breast cancer. The maintenance of mechanical conditioning is regulated by RUNX2, an osteogenic transcription factor, established driver of bone metastasis, and mitotic bookmarker that preserves chromatin accessibility at target gene loci. Using genetic and functional approaches, we demonstrate that mechanical conditioning maintenance can be simulated, repressed, or extended, with corresponding changes in bone metastatic potential.

Increasing passive compliance to wearing a facemask in children with autism spectrum disorder.

The current study taught 6 children with autism spectrum disorder (ASD) to increase passive compliance of wearing a facemask across sequentially increasing durations of time. A changing-criterion design embedded within a nonconcurrent multiple baseline design was used to evaluate the effectiveness of a resetting differential reinforcement of other behavior (DRO) without escape extinction procedure on passive compliance. Terminal probe sessions determined DRO fading intervals. Results showed that 2 participants acquired mastery level passive compliance (30 min) without fading during the initial baseline sessions. The remaining 4 participants acquired mastery level passive compliance following fading intervals within the DRO intervention. Participants' passive compliance generalized across 2 novel settings. This study replicates previous studies and extends empirical support for the use of DRO without escape extinction interventions for increasing passive compliance with medical devices in children with ASD.

When do errors in reinforcer delivery affect learning? A parametric analysis of treatment integrity.

Treatment integrity is the extent to which components of an intervention are implemented as intended (Gresham, 1989). Recent behavior-analytic literature has begun to evaluate the effects of reduced-treatment integrity on the efficacy and efficiency of skill-acquisition interventions. This study extended the current literature on the effects of errors of omission and commission of reinforcer delivery by replicating and extending Hirst and DiGennaro Reed (2015). Using a randomized-control group design, we compared undergraduate student participants' acquisition of conditional discriminations in a parametric analysis of different error values. A computer program erred in reinforcer delivery on 0%, 5%, 10%, 15%, 20%, 25% and 50% of trials. The purpose of the current study was to identify which levels of reduced integrity slowed or prevented acquisition. Our data replicated the findings of Hirst and DiGennaro Reed, and extended parametric analyses by identifying that errors in reinforcer delivery occurring on 15% or fewer trials (i.e., 85% integrity) were unlikely to prevent participants' responding from meeting the mastery criterion. These results could inform future research on how treatment-integrity errors change behavior-analytic procedures and the effects on skill acquisition for consumers of applied behavior analysis.

The Effects of Time Framing on Compliance to Hypothetical Social-Distancing Policies Related to COVID-19.

The current study analyzed the effects of two frames for durations of time-calendar unit and calendar date-on measures of compliance to hypothetical social-distancing policies related to the COVID-19 pandemic. Participants reported the extent to which they would comply with hypothetical social-distancing policies lasting different durations of time. Durations of time were framed as calendar units (e.g., days, weeks, months, years) and calendar dates (i.e., specific dates the policies would extent to). Levels of compliance across durations of time were used to calculate the area under the curve (AuC) for each condition. Social-distancing policies framed in calendar dates yielded significantly greater AuC values compared to social-distancing policies framed in calendar units. Participants' self-reported political affiliation yielded a significant main effect: Conservative participants' AuC values were significantly lower than liberal participants' AuC values. The framing of the duration of time was a significant variable in controlling rates of compliance to hypothetical social-distancing policies.

Effects of reward magnitude frames on measures of delay discounting in a hypothetical money scenario.

The current study analyzed the effects of three frames of reward magnitude-quantity, volume, and duration-on the rate at which college students discounted hypothetical, delayed monetary rewards. Hypothetical scenarios were presented using the fill-in-the-blank discounting questionnaire and participants made choices between immediate and delayed hypothetical monetary rewards. Scenarios framed the monetary choices as (a) quantity of dollar bills, (b) height (inches) of a stack of dollar bills, and (c) duration of time spent in a hypothetical cash machine to collect dollar bills. For each scenario, participants' subjective values were used to calculate the area under the curve (AuC). Framing resulted in a moderate effect size: The duration frame yielded significantly smaller AuC values compared to the quantity and volume frames. Thus, the framing of reward magnitude was a significant variable in controlling discounting rates for hypothetical, delayed monetary rewards. Subsequent investigations should be aware of the independent effects of the reward magnitude frames on delay discounting rates.

Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking.

Neutrophils are innate immune effector cells that traffic from the circulation to extravascular sites of inflammation. ß2 integrins are important mediators of the processes involved in neutrophil recruitment. Although neutrophils express the cytoskeletal protein vinculin, they do not form mature focal adhesions. Here, we characterize the role of vinculin in ß2 integrin-dependent neutrophil adhesion, migration, mechanosensing, and recruitment. We observe that knockout of vinculin attenuates, but does not completely abrogate, neutrophil adhesion, spreading, and crawling under static conditions. However, we also found that vinculin deficiency does not affect these behaviors in the presence of forces from fluid flow. In addition, we identify a role for vinculin in mechanosensing, as vinculin-deficient neutrophils exhibit attenuated spreading on stiff, but not soft, substrates. Consistent with these findings, we observe that in vivo neutrophil recruitment into the inflamed peritoneum of mice remains intact in the absence of vinculin. Together, these data suggest that while vinculin regulates some aspects of neutrophil adhesion and spreading, it may be dispensable for ß2 integrin-dependent neutrophil recruitment in vivo.

Measuring DNA content in live cells by fluorescence microscopy.

BACKGROUND: Live-cell fluorescence microscopy (LCFM) is a powerful tool used to investigate cellular dynamics in real time. However, the capacity to simultaneously measure DNA content in cells being tracked over time remains challenged by dye-associated toxicities. The ability to measure DNA content in single cells by means of LCFM would allow cellular stage and ploidy to be coupled with a variety of imaging directed analyses. Here we describe a widely applicable nontoxic approach for measuring DNA content in live cells by fluorescence microscopy. This method relies on introducing a live-cell membrane-permeant DNA fluorophore, such as Hoechst 33342, into the culture medium of cells at the end of any live-cell imaging experiment and measuring each cell's integrated nuclear fluorescence to quantify DNA content. Importantly, our method overcomes the toxicity and induction of DNA damage typically caused by live-cell dyes through strategic timing of adding the dye to the cultures; allowing unperturbed cells to be imaged for any interval of time before quantifying their DNA content. We assess the performance of our method empirically and discuss adaptations that can be implemented using this technique. RESULTS: Presented in conjunction with cells expressing a histone 2B-GFP fusion protein (H2B-GFP), we demonstrated how this method enabled chromosomal segregation errors to be tracked in cells as they progressed through cellular division that were later identified as either diploid or polyploid. We also describe and provide an automated Matlab-derived algorithm that measures the integrated nuclear fluorescence in each cell and subsequently plots these measurements into a cell cycle histogram for each frame imaged. The algorithm's accurate assessment of DNA content was validated by parallel flow cytometric studies. CONCLUSIONS: This method allows the examination of single-cell dynamics to be correlated with cellular stage and ploidy in a high-throughput fashion. The approach is suitable for any standard epifluorescence microscope equipped with a stable illumination source and either a stage-top incubator or an enclosed live-cell incubation chamber. Collectively, we anticipate that this method will allow high-resolution microscopic analysis of cellular processes involving cell cycle progression, such as checkpoint activation, DNA replication, and cellular division.

Examination of the Effects of Auditory and Textual Stimuli on Response Accuracy and Latency during a Math Task and Tangram Puzzle.

Although Skinner (1957) provided a behavioral account of verbal thinking, additional research is needed to evaluate stimuli that may influence covert verbal behavior that occurs between the onset of a verbal stimulus and the emission of a response during an episode of verbal thinking. The present investigation examined the effects of auditory distractors and/or textual stimuli during arithmetic problems and tangram puzzles on the participants' response latency and accuracy. In addition, we measured and categorized occurrences of vocal verbal behavior during the response interval. In Experiments 1 and 2, the experimenter played auditory distractors during a proportion of arithmetic problems. In Experiment 2, the experimenter also presented a textual stimulus of the arithmetic problem. In Experiment 3, the experimenter played auditory distractors during a proportion of tangram puzzles. Results showed that auditory distractors led to longer response latencies and reduced accuracy in Experiment 1. The addition of the textual stimulus during trials in Experiment 2 improved accuracy and reduced differences in response latency when the auditory distractors were and were not present during the response interval. The auditory distractors during tangram puzzles in Experiment 3 produced no differential effects on accuracy or latency to respond.

Vancomycin Reduces Cell Wall Stiffness and Slows Swim Speed of the Lyme Disease Bacterium.

Borrelia burgdorferi, the spirochete that causes Lyme disease, is a tick-transmitted pathogen that requires motility to invade and colonize mammalian and tick hosts. These bacteria use a unique undulating flat-wave shape to penetrate and propel themselves through host tissues. Previous mathematical modeling has suggested that the morphology and motility of these spirochetes depends crucially on the flagellar/cell wall stiffness ratio. Here, we test this prediction using the antibiotic vancomycin to weaken the cell wall. We found that low to moderate doses of vancomycin (≤2.0 µg/mL for 24 h) produced small alterations in cell shape and that as the dose was increased, cell speed decreased. Vancomycin concentrations >1.0 µg/mL also inhibited cell growth and led to bleb formation on a fraction of the cells. To quantitatively assess how vancomycin affects cell stiffness, we used optical traps to bend unflagellated mutants of B. burgdorferi. We found that in the presence of vancomycin, cell wall stiffness gradually decreased over time, with a 40% reduction in the bending stiffness after 36 h. Under the same conditions, the swimming speed of wild-type B. burgdorferi slowed by ∼15%, with only marginal changes to cell morphology. Interestingly, our biophysical model for the swimming dynamics of B. burgdorferi suggested that cell speed should increase with decreasing cell stiffness. We show that this discrepancy can be resolved if the periplasmic volume decreases as the cell wall becomes softer. These results provide a testable hypothesis for how alterations of cell wall stiffness affect periplasmic volume regulation. Furthermore, since motility is crucial to the virulence of B. burgdorferi, the results suggest that sublethal doses of antibiotics could negatively impact spirochete survival by impeding their swim speed, thereby enabling their capture and elimination by phagocytes.

The induction of endoreduplication and polyploidy by elevated expression of 14-3-3γ.

Several studies have demonstrated that specific 14-3-3 isoforms are frequently elevated in cancer and that these proteins play a role in human tumorigenesis. 14-3-3γ, an isoform recently demonstrated to function as an oncoprotein, is overexpressed in a variety of human cancers; however, its role in promoting tumorigenesis remains unclear. We previously reported that overexpression of 14-3-3γ caused the appearance of polyploid cells, a phenotype demonstrated to have profound tumor promoting properties. Here we examined the mechanism driving 14-3-3γ-induced polyploidization and the effect this has on genomic stability. Using FUCCI probes we showed that these polyploid cells appeared when diploid cells failed to enter mitosis and subsequently underwent endoreduplication. We then demonstrated that 14-3-3γ-induced polyploid cells experience significant chromosomal segregation errors during mitosis and observed that some of these cells stably propagate as tetraploids when isolated cells were expanded into stable cultures. These data lead us to conclude that overexpression of the 14-3-3γ promotes endoreduplication. We further investigated the role of 14-3-3γ in human NSCLC samples and found that its expression is significantly elevated in polyploid tumors. Collectively, these results suggests that 14-3-3γ may promote tumorigenesis through the production of a genetically unstable polyploid intermediate.

Viscous dynamics of Lyme disease and syphilis spirochetes reveal flagellar torque and drag.

The spirochetes that cause Lyme disease (Borrelia burgdorferi) and syphilis (Treponema pallidum) swim through viscous fluids, such as blood and interstitial fluid, by undulating their bodies as traveling, planar waves. These undulations are driven by rotation of the flagella within the periplasmic space, the narrow (∼20-40 nm in width) compartment between the inner and outer membranes. We show here that the swimming speeds of B. burgdorferi and T. pallidum decrease with increases in viscosity of the external aqueous milieu, even though the flagella are entirely intracellular. We then use mathematical modeling to show that the measured changes in speed are consistent with the exertion of constant torque by the spirochetal flagellar motors. Comparison of simulations, experiments, and a simple model for power dissipation allows us to estimate the torque and resistive drag that act on the flagella of these major spirochetal pathogens.

The heterogeneous motility of the Lyme disease spirochete in gelatin mimics dissemination through tissue.

The Lyme disease spirochete Borrelia burgdorferi exists in nature in an enzootic cycle that involves the arthropod vector Ixodes scapularis and mammalian reservoirs. To disseminate within and between these hosts, spirochetes must migrate through complex, polymeric environments such as the basement membrane of the tick midgut and the dermis of the mammal. To date, most research on the motility of B. burgdorferi has been done in media that do not resemble the tissue milieus that B. burgdorferi encounter in vivo. Here we show that the motility of Borrelia in gelatin matrices in vitro resembles the pathogen's movements in the chronically infected mouse dermis imaged by intravital microscopy. More specifically, B. burgdorferi motility in mouse dermis and gelatin is heterogeneous, with the bacteria transitioning between at least three different motility states that depend on transient adhesions to the matrix. We also show that B. burgdorferi is able to penetrate matrices with pore sizes much smaller than the diameter of the bacterium. We find a complex relationship between the swimming behavior of B. burgdorferi and the rheological properties of the gelatin, which cannot be accounted for by recent theoretical predictions for microorganism swimming in gels. Our results also emphasize the importance of considering borrelial adhesion as a dynamic rather than a static process.

The hybrid histidine kinase Hk1 is part of a two-component system that is essential for survival of Borrelia burgdorferi in feeding Ixodes scapularis ticks.

Two-component systems (TCS) are principal mechanisms by which bacteria adapt to their surroundings. Borrelia burgdorferi encodes only two TCS. One is comprised of a histidine kinase, Hk2, and the response regulator Rrp2. While the contribution of Hk2 remains unclear, Rrp2 is part of a regulatory pathway involving the spirochete's alternate sigma factors, RpoN and RpoS. Genes within the Rrp2/RpoN/RpoS regulon function to promote tick transmission and early infection. The other TCS consists of a hybrid histidine kinase, Hk1, and the response regulator Rrp1. Hk1 is composed of two periplasmic sensor domains (D1 and D2), followed by conserved cytoplasmic histidine kinase core, REC, and Hpt domains. In addition to its REC domain, Rrp1 contains a GGDEF motif characteristic of diguanylate cyclases. To investigate the role of Hk1 during the enzootic cycle, we inactivated this gene in two virulent backgrounds. Extensive characterization of the resulting mutants revealed a dramatic phenotype whereby Hk1-deficient spirochetes are virulent in mice and able to migrate out of the bite site during feeding but are killed within the midgut following acquisition. We hypothesize that the phosphorelay between Hk1 and Rrp1 is initiated by the binding of feeding-specific ligand(s) to Hk1 sensor domain D1 and/or D2. Once activated, Rrp1 directs the synthesis of cyclic dimeric GMP (c-di-GMP), which, in turn, modulates the expression and/or activity of gene products required for survival within feeding ticks. In contrast to the Rrp2/RpoN/RpoS pathway, which is active only within feeding nymphs, the Hk1/Rrp1 TCS is essential for survival during both larval and nymphal blood meals.