Single-molecule and super-resolved imaging deciphers membrane behavior of onco-immunogenic CCR5.

The ability of tumors to establish a pro-tumorigenic microenvironment is an important point of investigation in the search for new therapeutics. Tumors form microenvironments in part by the "education" of immune cells attracted via chemotactic axes such as that of CCR5-CCL5. Further, CCR5 upregulation by cancer cells, coupled with its association with pro-tumorigenic features such as drug resistance and metastasis, has suggested CCR5 as a therapeutic target. However, with several conformational "pools" being reported, phenotypic investigations must be capable of unveiling conformational heterogeneity. Addressing this challenge, we performed super-resolution structured illumination microscopy (SIM) and single molecule partially TIRF-coupled HILO (PaTCH) microscopy of CCR5 in fixed cells. SIM data revealed a non-random spatial distribution of CCR5 assemblies, while Intensity-tracking of CCR5 assemblies from PaTCH images indicated dimeric sub-units independent of CCL5 perturbation. These biophysical methods can provide important insights into the structure and function of onco-immunogenic receptors and many other biomolecules.

Exploring the Association Between Antibiotic Prescribing Patterns and Clinical Variations in a Network of Pediatricians.

INTRODUCTION: Variation in practice patterns among physicians is well-documented despite professional guidelines and increasingly uniform medical training. Variations may lead to improper utilization of healthcare resources, misdiagnosis, overdiagnosis, unnecessary treatments, and forgoing of needed interventions. One area of clinical variation and overuse of particular interest is the prescribing of antibiotics, which can lead to eventual antibiotic resistance and other negative consequences. Variations in antibiotic prescribing along with other practice patterns have been studied previously but no attempt has been made to examine the correlation between multiple practice patterns. The purpose of this study was to determine if a correlation existed between the provider behaviors studied. METHODS: A small area network of 39 pediatric providers was analyzed to determine if antibiotic prescription percentages varied. Antibiotic prescription percentages were further broken down by visit type (sick versus well). Two other practice measures, in-office lab utilization and diagnoses of food and drug allergies, were then analyzed. Data were explored primarily with Spearman's correlation tests. RESULTS: Strong positive correlation was seen between a provider's antibiotic prescription percentage at sick and well visits. Strong positive correlation was seen between the antibiotic prescribing percentage and the number of in-office labs ordered. Moderate positive correlation was seen between antibiotic prescribing percentage and the percentage of a provider's empaneled patients with any allergy diagnosis (medication, food, or seasonal). CONCLUSION: This retrospective study demonstrates that variation in provider practice patterns continues to exist despite established practice guidelines from national organizations. It also demonstrates a linear correlation between multiple provider behaviors that have not previously been explored together. The presence of a correlation between clinical behaviors may suggest an underlying practice philosophy and present an opportunity for personalized, provider-specific education and quality improvement.

Amyloid-ß oligomerization monitored by single-molecule stepwise photobleaching.

A major hallmark of Alzheimer's disease is the misfolding and aggregation of the amyloid- ß peptide (Aß). While early research pointed towards large fibrillar- and plaque-like aggregates as being the most toxic species, recent evidence now implicates small soluble Aß oligomers as being orders of magnitude more harmful. Techniques capable of characterizing oligomer stoichiometry and assembly are thus critical for a deeper understanding of the earliest stages of neurodegeneration and for rationally testing next-generation oligomer inhibitors. While the fluorescence response of extrinsic fluorescent probes such as Thioflavin-T have become workhorse tools for characterizing large Aß aggregates in solution, it is widely accepted that these methods suffer from many important drawbacks, including an insensitivity to oligomeric species. Here, we integrate several biophysics techniques to gain new insight into oligomer formation at the single-molecule level. We showcase single-molecule stepwise photobleaching of fluorescent dye molecules as a powerful method to bypass many of the traditional limitations, and provide a step-by-step guide to implementing the technique in vitro. By collecting fluorescence emission from single Aß(1-42) peptides labelled at the N-terminal position with HiLyte Fluor 555 via wide-field total internal reflection fluorescence (TIRF) imaging, we demonstrate how to characterize the number of peptides per single immobile oligomer and reveal heterogeneity within sample populations. Importantly, fluorescence emerging from Aß oligomers cannot be easily investigated using diffraction-limited optical microscopy tools. To assay oligomer activity, we also demonstrate the implementation of another biophysical method involving the ratiometric imaging of Fura-2-AM loaded cells which quantifies the rate of oligomer-induced dysregulation of intracellular Ca2+ homeostasis. We anticipate that the integrated single-molecule biophysics approaches highlighted here will develop further and in principle may be extended to the investigation of other protein aggregation systems under controlled experimental conditions.

Misery loves company: mood-congruent emotional responding to music.

We examined emotional responding to music after mood induction. On each trial, listeners heard a 30-s music excerpt and rated how much they liked it, whether it sounded happy or sad, and how familiar it was. When the excerpts sounded unambiguously happy or sad (Experiment 1), the typical preference for happy-sounding music was eliminated after inducing a sad mood. When the excerpts sounded ambiguous with respect to happiness and sadness (Experiment 2), listeners perceived more sadness after inducing a sad mood. Sad moods had no influence on familiarity ratings (Experiments 1 and 2). These findings imply that "misery loves company." Listeners in a sad mood fail to show the typical preference for happy-sounding music, and they perceive more sadness in music that is ambiguous with respect to mood.

Liking and identifying emotionally expressive music: age and gender differences.

Adults and children 5, 8, and 11 years of age listened to short excerpts of unfamiliar music that sounded happy, scary, peaceful, or sad. Listeners initially rated how much they liked each excerpt. They subsequently made a forced-choice judgment about the emotion that each excerpt conveyed. Identification accuracy was higher for young girls than for young boys, but both genders reached adult-like levels by age 11. High-arousal emotions (happiness and fear) were better identified than low-arousal emotions (peacefulness and sadness), and this advantage was exaggerated among younger children. Whereas children of all ages preferred excerpts depicting high-arousal emotions, adults favored excerpts depicting positive emotions (happiness and peacefulness). A preference for positive emotions over negative emotions was also evident among females of all ages. As identification accuracy improved, liking for positively valenced music increased among 5- and 8-year-olds but decreased among 11-year-olds.

The use of newborn screening pulse oximetry to detect cyanotic congenital heart disease: a survey of current practice at Army, Navy, and Air Force hospitals.

OBJECTIVE: To determine the prevalence of newborn screening pulse oximetry (+POx) among military hospitals, including barriers to instituting protocols. METHODS: An internet-based questionnaire was forwarded to the senior pediatricians at military hospitals worldwide supporting newborn deliveries. RESULTS: Forty seven of 53 hospitals (88%) supporting deliveries responded to the survey. Thirty percent of hospitals utilize a +POx protocol. Eight centers cited no problems with implementation. All hospitals screened at > or = 24 hours of life. The site of recording, positive values, and follow-up for positive screens varied. Cardiology consult and echocardiogram were not mandated. Most hospitals (34/47) are unable to obtain a pediatric cardiology consult without transfer. Few hospitals (9/47) utilize a telemedicine system. Seventy-five percent (24/32) of hospitals not utilizing a protocol are interested in instituting one. CONCLUSION: Though slightly less than one-third of military hospitals use a +POx, there is a greater interest in its use. More reliable consultative services and a robust telemedicine system may aid its implementation.