Clock-dependent chromatin topology modulates circadian transcription and behavior.

The circadian clock in animals orchestrates widespread oscillatory gene expression programs, which underlie 24-h rhythms in behavior and physiology. Several studies have shown the possible roles of transcription factors and chromatin marks in controlling cyclic gene expression. However, how daily active enhancers modulate rhythmic gene transcription in mammalian tissues is not known. Using circular chromosome conformation capture (4C) combined with sequencing (4C-seq), we discovered oscillatory promoter-enhancer interactions along the 24-h cycle in the mouse liver and kidney. Rhythms in chromatin interactions were abolished in arrhythmic Bmal1 knockout mice. Deleting a contacted intronic enhancer element in the Cryptochrome 1 (Cry1) gene was sufficient to compromise the rhythmic chromatin contacts in tissues. Moreover, the deletion reduced the daily dynamics of Cry1 transcriptional burst frequency and, remarkably, shortened the circadian period of locomotor activity rhythms. Our results establish oscillating and clock-controlled promoter-enhancer looping as a regulatory layer underlying circadian transcription and behavior.

Enhancement of dielectrophoresis-based particle collection from high conducting fluids due to partial electrode insulation.

Dielectrophoresis (DEP) is a successful method to recover nanoparticles from different types of fluid. The DEP force acting on these particles is created by an electrode microarray that produces a nonuniform electric field. To apply DEP to a highly conducting biological fluid, a protective hydrogel coating over the metal electrodes is required to create a barrier between the electrode and the fluid. This protects the electrodes, reduces the electrolysis of water, and allows the electric field to penetrate into the fluid sample. We observed that the protective hydrogel layer can separate from the electrode and form a closed domed structure and that collection of 100 nm polystyrene beads increased when this occurred. To better understand this collection increase, we used COMSOL Multiphysics software to model the electric field in the presence of the dome filled with different materials ranging from low-conducting gas to high conducting phosphate-buffered saline fluids. The results suggest that as the electrical conductivity of the material inside the dome is reduced, the whole dome acts as an insulator which increases electric field intensity at the electrode edge. This increased intensity widens the high-intensity electric field factor zone resulting in increased collection. This informs how dome formation results in increased particle collection and provides insight into how the electric field can be intensified to the increase collection of particles. These results have important applications for increasing the recovery of biologically-derived nanoparticles from undiluted physiological fluids that have high conductance, including the collection of cancer-derived extracellular vesicles from plasma for liquid biopsy applications.

cyc-DEP: Cyclic immunofluorescence profiling of particles collected using dielectrophoresis.

Cancer is a highly heterogenous disease that requires precise detection tools and active surveillance methods. Liquid biopsy assays provide an agnostic way to follow the complex trajectory of cancer, providing better patient stratification tools for optimized treatment. Here, we present the development of a low-volume liquid biopsy assay called cyc-DEP (cyclic immunofluorescent imaging on dielectrophoretic chip) to profile biomarkers collected on a dielectrophoretic microfluidic chip platform. To enable on-chip cyclic imaging, we optimized a fluorophore quenching method and sequential rounds of on-chip staining with fluorescently conjugated primary antibodies. cyc-DEP allows for the quantification of a multiplex array of proteins using 25 µl of a patient plasma sample. We utilized nanoparticles from a prostate adenocarcinoma (LNCaP) cell line and a panel of six target proteins to develop our proof-of-concept technique. We then used cyc-DEP to quantify blood plasma levels of target proteins from healthy individuals, low-grade and high-grade prostate cancer patients (n = 3 each) in order to demonstrate that our platform is suitable for liquid biopsy analysis in its present form. To ensure accurate quantification of signal intensities and comparisons between different samples, we incorporated a signal intensity normalization method (fluorescent beads) and a custom signal intensity quantification algorithm that account for the distribution of signal across hundreds of collection regions on each chip. Our technique enabled a threefold improvement in multiplicity for detecting proteins associated with fluid samples, opening doors for early detection, and active surveillance through quantification of a multiplex array of biomarkers from low-volume liquid biopsies.

Tracking progress towards malaria elimination in China: Individual-level estimates of transmission and its spatiotemporal variation using a diffusion network approach.

In order to monitor progress towards malaria elimination, it is crucial to be able to measure changes in spatio-temporal transmission. However, common metrics of malaria transmission such as parasite prevalence are under powered in elimination contexts. China has achieved major reductions in malaria incidence and is on track to eliminate, having reporting zero locally-acquired malaria cases in 2017 and 2018. Understanding the spatio-temporal pattern underlying this decline, especially the relationship between locally-acquired and imported cases, can inform efforts to maintain elimination and prevent re-emergence. This is particularly pertinent in Yunnan province, where the potential for local transmission is highest. Using a geo-located individual-level dataset of cases recorded in Yunnan province between 2011 and 2016, we introduce a novel Bayesian framework to model a latent diffusion process and estimate the joint likelihood of transmission between cases and the number of cases with unobserved sources of infection. This is used to estimate the case reproduction number, Rc. We use these estimates within spatio-temporal geostatistical models to map how transmission varied over time and space, estimate the timeline to elimination and the risk of resurgence. We estimate the mean Rc between 2011 and 2016 to be 0.171 (95% CI = 0.165, 0.178) for P. vivax cases and 0.089 (95% CI = 0.076, 0.103) for P. falciparum cases. From 2014 onwards, no cases were estimated to have a Rc value above one. An unobserved source of infection was estimated to be moderately likely (p>0.5) for 19/ 611 cases and high (p>0.8) for 2 cases, suggesting very high levels of case ascertainment. Our estimates suggest that, maintaining current intervention efforts, Yunnan is unlikely to experience sustained local transmission up to 2020. However, even with a mean of 0.005 projected up to 2020, locally-acquired cases are possible due to high levels of importation.

Utilization of High-Intensity Statins in Patients at Risk for Cardiovascular Events: A National Cross-Sectional Study.

BACKGROUND: The 2013 American College of Cardiology/American Heart Association cholesterol guidelines, which identified four groups of patients at risk for atherosclerotic cardiovascular disease events, departed from the target-based approach to managing cholesterol. The impact of these guidelines on high-intensity statin use across the United States is unclear. STUDY QUESTION: The primary objective was to evaluate the rate of high-intensity potential (HIP) statin use before and after the 2013 guidelines. The secondary objective was to identify predictors of HIP statin use within the study population. STUDY DESIGN: A national cross-sectional study was conducted using data from the National Ambulatory Medical Care Survey. Office visits involving patients aged 21-75 years where criteria for HIP statin therapy were met were included. Visits involving pregnant patients were excluded. MEASURES AND OUTCOMES: Prescribing trends of HIP statins were measured from National Ambulatory Medical Care Survey data before and after the 2013 guidelines. Multivariate logistic regression identified variables associated with prescribing HIP statins. RESULTS: A total of 48,884 visits were included, representing more than 940 million office visits nationally. HIP statins were listed in 9.5% and 16.5% of visits before and after 2013, respectively (odds ratio [OR] 1.88; 95% confidence interval [CI] 1.62-2.20). The strongest predictors of HIP statin use were antihypertensive use (OR 5.38, 95% CI 4.67-6.20), comorbid hyperlipidemia (OR 2.93, 95% CI 2.62-3.29), Black race (OR 0.63, 95% CI 0.49-0.81), and Hispanic ethnicity (OR 0.65, 95% CI 0.52-0.80). CONCLUSIONS: Prescribing rates for HIP statins increased after the release of the 2013 guidelines. The prescribing rates were lower than expected, especially in Black and Hispanic patients. These observations signify opportunities to improve the quality of care for patients who are at risk for atherosclerotic cardiovascular disease events in the United States.

Image method for electrostatic energy of polarizable dipolar spheres.

The multiple-scattering theory for the electrostatics of many-body systems of monopolar spherical particles, embedded in a dielectric medium, is generalized to describe the electrostatics of these particles with embedded dipoles and multipoles. The Neumann image line construction for the electrostatic polarization produced by one particle is generalized to compute the energy, forces, and torques for the many-body system as functions of the positions of the particles. The approach is validated by comparison with direct numerical calculation, and the convergence rate is analyzed and expressed in terms of the discontinuity in dielectric contrast and particle density. As an illustration of this formalism, the stability of small particle clusters is analyzed. The theory is developed in a form that can readily be adapted to Monte Carlo and molecular dynamics simulations for polarizable particles and, more generally, to study the interactions among polarizable molecules.

Fluctuation-Driven Neural Dynamics Reproduce Drosophila Locomotor Patterns.

The neural mechanisms determining the timing of even simple actions, such as when to walk or rest, are largely mysterious. One intriguing, but untested, hypothesis posits a role for ongoing activity fluctuations in neurons of central action selection circuits that drive animal behavior from moment to moment. To examine how fluctuating activity can contribute to action timing, we paired high-resolution measurements of freely walking Drosophila melanogaster with data-driven neural network modeling and dynamical systems analysis. We generated fluctuation-driven network models whose outputs-locomotor bouts-matched those measured from sensory-deprived Drosophila. From these models, we identified those that could also reproduce a second, unrelated dataset: the complex time-course of odor-evoked walking for genetically diverse Drosophila strains. Dynamical models that best reproduced both Drosophila basal and odor-evoked locomotor patterns exhibited specific characteristics. First, ongoing fluctuations were required. In a stochastic resonance-like manner, these fluctuations allowed neural activity to escape stable equilibria and to exceed a threshold for locomotion. Second, odor-induced shifts of equilibria in these models caused a depression in locomotor frequency following olfactory stimulation. Our models predict that activity fluctuations in action selection circuits cause behavioral output to more closely match sensory drive and may therefore enhance navigation in complex sensory environments. Together these data reveal how simple neural dynamics, when coupled with activity fluctuations, can give rise to complex patterns of animal behavior.

Atrazine reduces the transmission of an amphibian trematode by altering snail and ostracod host-parasite interactions.

Trematodes are ubiquitous members of aquatic environments, have many functional roles in ecosystems, and can cause diseases in humans, livestock, and wild animals. Despite their importance and reports of parasite population declines, few studies have concurrently assessed the effects of aquatic contaminants on multiple hosts, multiple parasite life cycle stages, and on transmission-related host-parasite interactions. Here, we test the effects of environmentally relevant concentrations of the herbicide atrazine (0, 3, 30 µg/L) on the establishment and development of an amphibian trematode (Halipegus eccentricus) in a first-intermediate snail host (Physa acuta) and in a second-intermediate ostracod host (Cypridopsis sp.). Additionally, we test the interactive effects of atrazine and parasitism on snail and ostracod survival. Our results indicate that atrazine negatively affects trematode transmission by altering snail and ostracod host-parasite interactions. Although atrazine did not affect the survival of uninfected snails alone, atrazine acted synergistically with parasitism to reduce the longevity of infected snails. As a result, the number of cercariae (i.e., larval trematodes) produced by snails was 50.7 % (3 µg/L) and 14.9 % (30 µg/L) relative to controls. Atrazine exhibited direct negative effects on ostracod survival at 30 µg/L. However, when ostracods were also exposed to trematodes, the negative effects of atrazine on survival were diminished. Although infected ostracod survival remained high, trematode development was significantly reduced, resulting in reduced infectivity of metacercariae (i.e., nongravid adult cysts infective to definite host) to 32.2 % (3 µg/L) and 28.6 % (30 µg/L) relative to the controls. The combination of reduced cercaria production and reduced metacercarial infectivity in the 3 and 30 µg/L atrazine treatment groups reduced the net number of infective worms produced to 16.4 and 4.3 % (respectively) relative to the control. These results demonstrate the complex nature of pesticide effects on trematode infections and indicate that trematodes can affect their first- and second-intermediate hosts differently under different pesticide concentrations. Our work has broad implications for parasite transmission and conservation and provides a testable mechanism for understanding trematode population declines in contaminated wetlands.

Robust synchronization of coupled circadian and cell cycle oscillators in single mammalian cells.

Circadian cycles and cell cycles are two fundamental periodic processes with a period in the range of 1 day. Consequently, coupling between such cycles can lead to synchronization. Here, we estimated the mutual interactions between the two oscillators by time-lapse imaging of single mammalian NIH3T3 fibroblasts during several days. The analysis of thousands of circadian cycles in dividing cells clearly indicated that both oscillators tick in a 1:1 mode-locked state, with cell divisions occurring tightly 5 h before the peak in circadian Rev-Erbα-YFP reporter expression. In principle, such synchrony may be caused by either unidirectional or bidirectional coupling. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling unambiguously show that the reverse coupling is predominant in NIH3T3 cells. Moreover, temperature, genetic, and pharmacological perturbations showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer.

The effects of the herbicide atrazine on freshwater snails.

Atrazine has been shown to affect freshwater snails from the subcellular to community level. However, most studies have used different snail species, methods, endpoints, and atrazine exposure concentrations, resulting in some conflicting results and limiting our understanding. The goal of this study was to address these concerns by (1) investigating the acute and chronic effects of atrazine on four species of freshwater snails (Biomphalaria glabrata, Helisoma trivolvis, Physa acuta, and Stagnicola elodes) using the same methods, endpoints, and concentrations, and (2) summarizing the current literature pertaining to the effects of atrazine on freshwater snails. We conducted a 48 h acute toxicity test with an atrazine concentration higher than what typically occurs in aquatic environments (1000 µg/L). Additionally, we exposed snails to environmentally relevant atrazine concentrations (0, 0.3, 3, and 30 µg/L) for 28 days and assessed snail survival, growth, and reproduction. We also summarized all known literature pertaining to atrazine effects on freshwater snails. The literature summary suggests snails are often affected by environmentally relevant atrazine concentrations at the subcellular and cellular levels. These effects are typically not transitive to effects on survival, growth, or reproduction at the same concentrations. Our acute exposures corroborate the general trend of no direct effect on snail populations as atrazine did not directly affect the survival of any of the four snail species. Similarly, environmentally relevant concentrations did not significantly affect the survival, growth, or reproduction of any snail species. These results indicate that, in the absence of other possible stressors, the direct effects of environmentally relevant atrazine concentrations may not be realized at the snail population level.

THE COCCIDIA SPECIES PFEIFFERINELLA ELLIPSOIDES EXHIBITS SPATIAL AND SPECIES-SPECIFIC VARIATION IN PREVALENCE AMONG FRESHWATER SNAILS.

Freshwater snails are commonly studied within the context of their role as intermediate hosts for digenetic trematodes. However, there are fundamental data deficiencies related to our understanding of directly transmitted parasites, such as coccidia, for freshwater snails. Because variation in coccidia pathogenicity and transmission among snail species likely has major impacts on snail community structure, we aimed to investigate the spatial distribution and prevalence of coccidia in several freshwater snail species throughout the Ozark and Ouachita Mountains ecoregions in Arkansas. We opportunistically collected 220 freshwater snails from 24 Ozark sites in summer 2022 and scanned fecal slides for the presence of coccidia. In summer 2023, we surveyed an additional 146 snails from 19 Ouachita sites. To test for apparent interactions among coccidia and trematodes, we scanned feces from a subset of snails (Physa and Planorbella in the Ozarks) that did not have concurrent trematode infections and from those that did. We observed oocysts that morphologically conformed to Pfeifferinella ellipsoides in 2 of the 9 snail taxa from 7 of the 43 sites. Planorbella trivolvis was infected at 2 of 6 sites in the Ozarks and 0 of 5 sites in the Ouachitas. Physa species were infected at 6 of 14 sites in the Ozarks and 0 of 12 sites in the Ouachitas. In the Ozarks, Pl. trivolvis had an overall prevalence of 0.13 (6 of 47), whereas individuals in the genus Physa had an overall prevalence of 0.08 (8 of 97). Our chi-square and Fisher exact tests revealed no significant evidence for trematode-coccidia competition or synergism within the two snail species. There were no other species infected, and we did not observe any coccidia in the snails from the Ouachitas. Our survey of 366 snails among 9 taxa and 43 sites represents the largest survey for freshwater snail coccidia to date and indicates that both Pl. trivolvis and Physa spp. may be primary hosts and/or reservoir hosts for Pf. ellipsoides in freshwater snail communities. The highly aggregated distribution of Pf. ellipsoides in northwestern Arkansas requires further investigation. Our results led to proposal of several hypotheses for additional research, including questions regarding the variation of coccidia host specificity and virulence.

A scoping review of artificial intelligence within pharmacy education.

OBJECTIVES: This scoping review aimed to summarize the available literature on the use of artificial intelligence (AI) in pharmacy education and identify gaps where additional research is needed. FINDINGS: Seven studies specifically addressing the use of AI in pharmacy education were identified. Of these 7 studies, 5 focused on AI use in the context of teaching and learning, 1 on the prediction of academic performance for admissions, and the final study focused on using AI text generation to elucidate the benefits and limitations of ChatGPT use in pharmacy education. SUMMARY: There are currently a limited number of available publications that describe AI use in pharmacy education. Several challenges exist regarding the use of AI in pharmacy education, including the need for faculty expertise and time, limited generalizability of tools, limited outcomes data, and several legal and ethical concerns. As AI use increases and implementation becomes more standardized, opportunities will be created for the inclusion of AI in pharmacy education.

Concurrent validity of the Professional Fulfillment Index in a sample of pharmacy residents and preceptors.

PURPOSE: The purpose of this study was to validate the Stanford Professional Fulfillment Index (PFI) for assessment of burnout and professional fulfillment in a study population of pharmacy residents and residency preceptors. SUMMARY: The historical gold standard for assessing professional burnout is the Maslach Burnout Inventory (MBI); there is no established standard for professional fulfillment. The PFI is a 16-question assessment that has previously been validated in medical residents and practicing physicians. In this study, surveys including both PFI and MBI items were sent to active pharmacy residents and residency preceptors. To determine concurrent validity, domains of the PFI were compared to the closest related MBI domain as well as composite burnout rates measured in each portion of the survey. A total of 142 preceptors and 68 residents completed both the PFI and a version of the MBI previously validated in physicians. In assessing indicators of pharmacist burnout and fulfillment, data captured by domains of the PFI closely correlated with data captured by corresponding domains of the MBI (Pearson correlations of 0.683-0.822), with high internal consistency (Cronbach α of 0.866-0.903). CONCLUSION: The PFI is a valid method of assessing burnout in both pharmacy residents and residency preceptors. Additionally, the PFI contributes a reliable system of assessing professional fulfillment while also being highly accessible for both research and residency program monitoring applications.

SEASONAL OCCURRENCE OF NEOECHINORHYNCHUS EMYDIS (PHYLUM: ACANTHOCEPHALA) IN THE FRESHWATER SNAIL, PLANORBELLA CF. P. TRIVOLVIS, FROM OKLAHOMA.

The acanthocephalan Neoechinorhynchus emydis has a complex life cycle and infects turtle, ostracod, and snail hosts. However, little information is available on the seasonal distribution or the effects of N. emydis on freshwater snail hosts. To address this, we examined the seasonal distribution and melanization of acanthocephalans in Planorbella cf. Planorbella trivolvis snails from a single location in north-central Oklahoma. Seasonally, prevalence of N. emydis was 0% during the winter, increased to 50% during the summer, and declined to 17% in the fall. Mean abundance exhibited more variation but generally followed a similar pattern as prevalence. More important, all acanthocephalans located within the head/foot region of snail hosts contained melaninlike pigment surrounding each worm, suggesting that snails were mounting an immunological reaction to infections with N. emydis. Snail shell diameter was greatest during the fall and decreased during the winter, indicating that larger or older snails were dying during the winter. However, because field-collected snails were commonly infected with trematodes, and snail size varied significantly with season, it was unclear whether the observed seasonal dynamics of acanthocephalan infections were a result of snail mortality resulting from snail age, parasitic infections, or a combination of factors. To control for these factors, we exposed laboratory-reared Planorbella cf. P. trivolvis snails to naturally infected ostracods in field cages for 5-wk intervals during the winter, spring, and summer. Data from snail-cage infections were consistent with the seasonal field survey such that N. emydis infections were highest in the summer (20%) and lowest (0%) in the winter, suggesting that snails were not ingesting infected ostracods during the winter. However, fewer of our laboratory-reared snails survived in field cages during winter than during spring and summer, suggesting that snails may die more often during harsh winter conditions. Finally, we conducted a laboratory survival experiment by testing the life span and egg production of field-collected snails of various sizes that were naturally infected with acanthocephalans or trematodes or both. Our snail-survival experiment indicated that snail size but not infection status with acanthocephalans or trematodes affected snail survival, with larger snails surviving a shorter amount of time than smaller snails. In addition, snails infected with trematodes laid significantly fewer eggs compared with uninfected snails or snails infected with acanthocephalans. However, we found no significant difference in the number of eggs laid by acanthocephalan-infected and uninfected snails. Although other abiotic factors still need evaluation, we suggest that the occurrence of acanthocephalans in snails throughout the year may be partially influenced by the abundance of infected ostracods that snails may be ingesting and snail population fluctuations during the year.

Modeling PFAS Fate and Transport in Groundwater, with and Without Precursor Transformation.

Groundwater professionals require tools to evaluate a variety of technical issues related to per- and polyfluoroalkyl substances (PFAS). These include the potential impact of PFAS precursors on groundwater plumes of perfluoroalkyl acids (PFAAs). Numerical modeling results show that, by adjusting the mass loading rate, source zones with or without a precursor can produce similar PFAA plumes. However, if a precursor is present, it can impact PFAA plume concentrations and extend PFAA plume durations by decades. Additional research regarding in situ precursor transformation rates-and improvements in source area characterization-will further advance the predictive value of modeling.

Multi-population puma connectivity could restore genomic diversity to at-risk coastal populations in California.

Urbanization is decreasing wildlife habitat and connectivity worldwide, including for apex predators, such as the puma (Puma concolor). Puma populations along California's central and southern coastal habitats have experienced rapid fragmentation from development, leading to calls for demographic and genetic management. To address urgent conservation genomic concerns, we used double-digest restriction-site associated DNA (ddRAD) sequencing to analyze 16,285 genome-wide single-nucleotide polymorphisms (SNPs) from 401 pumas sampled broadly across the state. Our analyses indicated support for 4-10 geographically nested, broad- to fine-scale genetic clusters. At the broadest scale, the four genetic clusters had high genetic diversity and exhibited low linkage disequilibrium, indicating that pumas have retained genomic diversity statewide. However, multiple lines of evidence indicated substructure, including 10 finer-scale genetic clusters, some of which exhibited fixed alleles and linkage disequilibrium. Fragmented populations along the Southern Coast and Central Coast had particularly low genetic diversity and strong linkage disequilibrium, indicating genetic drift and close inbreeding. Our results demonstrate that genetically at risk populations are typically nested within a broader-scale group of interconnected populations that collectively retain high genetic diversity and heterogenous fixations. Thus, extant variation at the broader scale has potential to restore diversity to local populations if management actions can enhance vital gene flow and recombine locally sequestered genetic diversity. These state- and genome-wide results are critically important for science-based conservation and management practices. Our nested population genomic analysis highlights the information that can be gained from population genomic studies aiming to provide guidance for the conservation of fragmented populations.

Hybrid Silica-Coated PLGA Nanoparticles for Enhanced Enzyme-Based Therapeutics.

Some cancer cells rely heavily on non-essential biomolecules for survival, growth, and proliferation. Enzyme based therapeutics can eliminate these biomolecules, thus specifically targeting neoplastic cells; however, enzyme therapeutics are susceptible to immune clearance, exhibit short half-lives, and require frequent administration. Encapsulation of therapeutic cargo within biocompatible and biodegradable poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) is a strategy for controlled release. Unfortunately, PLGA NPs exhibit burst release of cargo shortly after delivery or upon introduction to aqueous environments where they decompose via hydrolysis. Here, we show the generation of hybrid silica-coated PLGA (SiLGA) NPs as viable drug delivery vehicles exhibiting sub-200 nm diameters, a metastable Zeta potential, and high loading efficiency and content. Compared to uncoated PLGA NPs, SiLGA NPs offer greater retention of enzymatic activity and slow the burst release of cargo. Thus, SiLGA encapsulation of therapeutic enzymes, such as asparaginase, could reduce frequency of administration, increase half-life, and improve efficacy for patients with a range of diseases.

Multicenter Retrospective Review of Ketamine Use in the ICU.

The response of ICU patients to continuously infused ketamine when it is used for analgesia and/or sedation remains poorly established. OBJECTIVES: To describe continuous infusion (CI) ketamine use in critically ill patients, including indications, dose and duration, adverse effects, patient outcomes, time in goal pain/sedation score range, exposure to analgesics/sedatives, and delirium. DESIGN SETTING AND PARTICIPANTS: Multicenter, retrospective, observational study from twenty-five diverse institutions in the United States. Patients receiving CI ketamine between January 2014 and December 2017. MAIN OUTCOMES AND MEASURES: Chart review evaluating institutional and patient demographics, ketamine indication, dose, administration, and adverse effects. Pain/sedation scores, cumulative doses of sedatives and analgesics, and delirium screenings in the 24 hours prior to ketamine were compared with those at 0-24 hours and 25-48 hours after. RESULTS: A total of 390 patients were included (median age, 54.5 yr; interquartile range, 39-65 yr; 61% males). Primary ICU types were medical (35.3%), surgical (23.3%), and trauma (17.7%). Most common indications were analgesia/sedation (n = 357, 91.5%). Starting doses were 0.2 mg/kg/hr (0.1-0.5 mg/kg/hr) and continued for 1.6 days (0.6-2.9 d). Hemodynamics in the first 4 hours after ketamine were variable (hypertension 24.0%, hypotension 23.5%, tachycardia 19.5%, bradycardia 2.3%); other adverse effects were minimal. Compared with 24 hours prior, there was a significant increase in proportion of time spent within goal pain score after ketamine initiation (24 hr prior: 68.9% [66.7-72.6%], 0-24 hr: 78.6% [74.3-82.5%], 25-48 hr: 80.3% [74.6-84.3%]; p < 0.001) and time spent within goal sedation score (24 hr prior: 57.1% [52.5-60.0%], 0-24 hr: 64.1% [60.7-67.2%], 25-48 hr: 68.9% [65.5-79.5%]; p < 0.001). There was also a significant reduction in IV morphine (mg) equivalents (24 hr prior: 120 [25-400], 0-24 hr: 118 [10-363], 25-48 hr: 80 [5-328]; p < 0.005), midazolam (mg) equivalents (24 hr prior: 11 [4-67], 0-24 hr: 6 [0-68], 25-48 hr: 3 [0-57]; p < 0.001), propofol (mg) (24 hr prior: 942 [223-4,018], 0-24 hr: 160 [0-2,776], 25-48 hr: 0 [0-1,859]; p < 0.001), and dexmedetomidine (µg) (24 hr prior: 1,025 [276-1,925], 0-24 hr: 285 [0-1,283], 25-48 hr: 0 [0-826]; p < 0.001). There was no difference in proportion of time spent positive for delirium (24 hr prior: 43.0% [17.0-47.0%], 0-24 hr: 39.5% [27.0-43.8%], 25-48 hr: 0% [0-43.7%]; p = 0.233). Limitations to these data include lack of a comparator group, potential for confounders and selection bias, and varying pain and sedation practices that may have changed since completion of the study. CONCLUSIONS AND RELEVANCE: There is variability in the use of CI ketamine. Hemodynamic instability was the most common adverse effect. In the 48 hours after ketamine initiation compared with the 24 hours prior, proportion of time spent in goal pain/sedation score range increased and exposure to other analgesics/sedatives decreased.

Label-free enrichment of rare unconventional circulating neoplastic cells using a microfluidic dielectrophoretic sorting device.

Cellular circulating biomarkers from the primary tumor such as circulating tumor cells (CTCs) and circulating hybrid cells (CHCs) have been described to harbor tumor-like phenotype and genotype. CHCs are present in higher numbers than CTCs supporting their translational potential. Methods for isolation of CHCs do not exist and are restricted to low-throughput, time consuming, and biased methodologies. We report the development of a label-free dielectrophoretic microfluidic platform facilitating enrichment of CHCs in a high-throughput and rapid fashion by depleting healthy peripheral blood mononuclear cells (PBMCs). We demonstrated up to 96.5% depletion of PBMCs resulting in 18.6-fold enrichment of cancer cells. In PBMCs from pancreatic adenocarcinoma patients, the platform enriched neoplastic cells identified by their KRAS mutant status using droplet digital PCR with one hour of processing. Enrichment was achieved in 75% of the clinical samples analyzed, establishing this approach as a promising way to non-invasively analyze tumor cells from patients.

Automated fluorescence quantification of extracellular vesicles collected from blood plasma using dielectrophoresis.

Tumor-secreted exosomes and other extracellular vesicles (EVs) in circulation contain valuable biomarkers for early cancer detection and screening. We have previously demonstrated collection of cancer-derived nanoparticles (NPs) directly from whole blood and plasma with a chip-based technique that uses a microelectrode array to generate dielectrophoretic (DEP) forces. This technique enables direct recovery of NPs from whole blood and plasma. The biomarker payloads associated with collected particles can be detected and quantified with immunostaining. Accurately separating the fluorescence intensity of stained biomarkers from background (BG) levels becomes a challenge when analyzing the blood from early-stage cancer patients in which biomarker concentrations are low. To address this challenge, we developed two complementary techniques to standardize the quantification of fluorescently immunolabeled biomarkers collected and concentrated at predictable locations within microfluidic chips. The first technique was an automated algorithm for the quantitative analysis of fluorescence intensity at collection regions within the chip compared to levels at adjacent regions. The algorithm used predictable locations of particle collection within the chip geometry to differentiate regions of collection and BG. We successfully automated the identification and removal of optical artifacts from quantitative calculations. We demonstrated that the automated system performs nearly the same as a human user following a standard protocol for manual artifact removal with Pearson's r-values of 0.999 and 0.998 for two different biomarkers (n = 36 patients). We defined a usable dynamic range of fluorescence intensities corresponding to 1 to 2000 arbitrary units (a.u.). Fluorescence intensities within the dynamic range increased linearly with respect to exposure time and particle concentration. The second technique was the implementation of an internal standard to adjust levels of biomarker fluorescence based on the relative collection efficiency of the chip. Use of the internal standard reduced variability in measured biomarker levels due to differences in chip-to-chip collection efficiency, especially at low biomarker concentrations. The internal standard did not affect linear trends between fluorescence intensity and exposure time. Adjustments using the internal standard improved linear trends between fluorescence intensity and particle concentration. The optical quantification techniques described in this paper can be easily adapted for other lab-on-a-chip platforms that have predefined regions of biomarker or particle collection and that rely on fluorescence detection.