Systematic replication enables normalization of high-throughput imaging assays.

MOTIVATION: High-throughput fluorescent microscopy is a popular class of techniques for studying tissues and cells through automated imaging and feature extraction of hundreds to thousands of samples. Like other high-throughput assays, these approaches can suffer from unwanted noise and technical artifacts that obscure the biological signal. In this work, we consider how an experimental design incorporating multiple levels of replication enables the removal of technical artifacts from such image-based platforms. RESULTS: We develop a general approach to remove technical artifacts from high-throughput image data that leverages an experimental design with multiple levels of replication. To illustrate the methods, we consider microenvironment microarrays (MEMAs), a high-throughput platform designed to study cellular responses to microenvironmental perturbations. In application to MEMAs, our approach removes unwanted spatial artifacts and thereby enhances the biological signal. This approach has broad applicability to diverse biological assays. AVAILABILITY AND IMPLEMENTATION: Raw data are on synapse (syn2862345), analysis code is on github: gjhunt/mema_norm, a reproducible Docker image is available on dockerhub: gjhunt/mema_norm. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Automatic Transformation and Integration to Improve Visualization and Discovery of Latent Effects in Imaging Data.

Proper data transformation is an essential part of analysis. Choosing appropriate transformations for variables can enhance visualization, improve efficacy of analytical methods, and increase data interpretability. However determining appropriate transformations of variables from high-content imaging data poses new challenges. Imaging data produces hundreds of covariates from each of thousands of images in a corpus. Each of these covariates will have a different distribution and need a potentially different transformation. As such imaging data produces hundreds of covariates, determining an appropriate transformation for each of them is infeasible by hand. In this paper we explore simple, robust, and automatic transformations of high-content image data. A central application of our work is to microenvironment microarray bio-imaging data from the NIH LINCS program. We show that our robust transformations enhance visualization and improve the discovery of substantively relevant latent effects. These transformations enhance analysis of image features individually and also improve data integration approaches when combining together multiple features. We anticipate that the advantages of this work will likely also be realized in the analysis of data from other high-content and highly-multiplexed technologies like Cell Painting or Cyclic Immunofluorescence. Software and further analysis can be found at gjhunt.github.io/rr.

dtangle: accurate and robust cell type deconvolution.

MOTIVATION: Cell type composition of tissues is important in many biological processes. To help understand cell type composition using gene expression data, methods of estimating (deconvolving) cell type proportions have been developed. Such estimates are often used to adjust for confounding effects of cell type in differential expression analysis (DEA). RESULTS: We propose dtangle, a new cell type deconvolution method. dtangle works on a range of DNA microarray and bulk RNA-seq platforms. It estimates cell type proportions using publicly available, often cross-platform, reference data. We evaluate dtangle on 11 benchmark datasets showing that dtangle is competitive with published deconvolution methods, is robust to outliers and selection of tuning parameters, and is fast. As a case study, we investigate the human immune response to Lyme disease. dtangle's estimates reveal a temporal trend consistent with previous findings and are important covariates for DEA across disease status. AVAILABILITY AND IMPLEMENTATION: dtangle is on CRAN (cran.r-project.org/package=dtangle) or github (dtangle.github.io). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Saturn's magnetic field revealed by the Cassini Grand Finale.

During 2017, the Cassini fluxgate magnetometer made in situ measurements of Saturn's magnetic field at distances ~2550 ± 1290 kilometers above the 1-bar surface during 22 highly inclined Grand Finale orbits. These observations refine the extreme axisymmetry of Saturn's internal magnetic field and show displacement of the magnetic equator northward from the planet's physical equator. Persistent small-scale magnetic structures, corresponding to high-degree (>3) axisymmetric magnetic moments, were observed. This suggests secondary shallow dynamo action in the semiconducting region of Saturn's interior. Some high-degree magnetic moments could arise from strong high-latitude concentrations of magnetic flux within the planet's deep dynamo. A strong field-aligned current (FAC) system is located between Saturn and the inner edge of its D-ring, with strength comparable to the high-latitude auroral FACs.

Myocardial Reparative Properties of Cardiac Mesenchymal Cells Isolated on the Basis of Adherence.

BACKGROUND: The authors previously reported that the c-kit-positive (c-kitPOS) cells isolated from slowly adhering (SA) but not from rapidly adhering (RA) fractions of cardiac mesenchymal cells (CMCs) are effective in preserving left ventricular (LV) function after myocardial infarction (MI). OBJECTIVES: This study evaluated whether adherence to plastic alone, without c-kit sorting, was sufficient to isolate reparative CMCs. METHODS: RA and SA CMCs were isolated from mouse hearts, expanded in vitro, characterized, and evaluated for therapeutic efficacy in mice subjected to MI. RESULTS: Morphological and phenotypic analysis revealed that murine RA and SA CMCs are indistinguishable; nevertheless, transcriptome analysis showed that they possess fundamentally different gene expression profiles related to factors that regulate post-MI LV remodeling and repair. A similar population of SA CMCs was isolated from porcine endomyocardial biopsy samples. In mice given CMCs 2 days after MI, LV ejection fraction 28 days later was significantly increased in the SA CMC group (31.2 ± 1.0% vs. 24.7 ± 2.2% in vehicle-treated mice; p < 0.05) but not in the RA CMC group (24.1 ± 1.2%). Histological analysis showed reduced collagen deposition in the noninfarcted region in mice given SA CMCs (7.6 ± 1.5% vs. 14.5 ± 2.8% in vehicle-treated mice; p < 0.05) but not RA CMCs (11.7 ± 1.7%), which was associated with reduced infiltration of inflammatory cells (14.1 ± 1.6% vs. 21.3 ± 1.5% of total cells in vehicle and 19.3 ± 1.8% in RA CMCs; p < 0.05). Engraftment of SA CMCs was negligible, which implies a paracrine mechanism of action. CONCLUSIONS: We identified a novel population of c-kit-negative reparative cardiac cells (SA CMCs) that can be isolated with a simple method based on adherence to plastic. SA CMCs exhibited robust reparative properties and offered numerous advantages, appearing to be more suitable than c-kitPOS cardiac progenitor cells for widespread clinical therapeutic application.

Effects of Intracoronary Infusion of Escalating Doses of Cardiac Stem Cells in Rats With Acute Myocardial Infarction.

BACKGROUND: Although c-kit(pos) cardiac stem cells (CSCs) preserve left ventricular (LV) function and structure after myocardial infarction, CSC doses have been chosen arbitrarily, and the dose-effect relationship is unknown. METHODS AND RESULTS: Rats underwent a 90-minute coronary occlusion followed by 35 days of reperfusion. Vehicle or CSCs at 5 escalating doses (0.3×10(6), 0.75×10(6), 1.5×10(6), 3.0×10(6), and 6.0×10(6) cells/heart) were given intracoronarily 4 h after reperfusion. The lowest dose (0.3×10(6)) had no effect on LV function and morphology, whereas 0.75, 1.5, and 3.0×10(6) significantly improved regional and global LV function (echocardiography and hemodynamic studies). These 3 doses had similar effects on echocardiographic parameters (infarct wall thickening fraction, LV end-systolic and end-diastolic volumes, LV ejection fraction) and hemodynamic variables (LV end-diastolic pressure, LV dP/dtmax, preload adjusted maximal power, end-systolic elastance, preload recruitable stroke work) and produced similar reductions in apoptosis, scar size, infarct wall thinning, and LV expansion index and similar increases in viable myocardium in the risk region (morphometry). Infusion of 6.0×10(6) CSCs markedly increased postprocedural mortality. Green fluorescent protein and 5-bromo-2'-deoxyuridine staining indicated that persistence of donor cells and formation of new myocytes were negligible with all doses. CONCLUSIONS: Surprisingly, in this rat model of acute myocardial infarction, the dose-response relationship for intracoronary CSCs is flat. A minimal dose between 0.3 and 0.75×10(6) is necessary for efficacy; above this threshold, a 4-fold increase in cell number does not produce greater improvement in LV function or structure. Further increases in cell dose are harmful.

Safety of intracoronary infusion of 20 million C-kit positive human cardiac stem cells in pigs.

BACKGROUND: There is mounting interest in using c-kit positive human cardiac stem cells (c-kit(pos) hCSCs) to repair infarcted myocardium in patients with ischemic cardiomyopathy. A recent phase I clinical trial (SCIPIO) has shown that intracoronary infusion of 1 million hCSCs is safe. Higher doses of CSCs may provide superior reparative ability; however, it is unknown if doses >1 million cells are safe. To address this issue, we examined the effects of 20 million hCSCs in pigs. METHODS: Right atrial appendage samples were obtained from patients undergoing cardiac surgery. The tissue was processed by an established protocol with eventual immunomagnetic sorting to obtain in vitro expanded hCSCs. A cumulative dose of 20 million cells was given intracoronarily to pigs without stop flow. Safety was assessed by measurement of serial biomarkers (cardiac: troponin I and CK-MB, renal: creatinine and BUN, and hepatic: AST, ALT, and alkaline phosphatase) and echocardiography pre- and post-infusion. hCSC retention 30 days after infusion was quantified by PCR for human genomic DNA. All personnel were blinded as to group assignment. RESULTS: Compared with vehicle-treated controls (n=5), pigs that received 20 million hCSCs (n=9) showed no significant change in cardiac function or end organ damage (assessed by organ specific biomarkers) that could be attributed to hCSCs (P>0.05 in all cases). No hCSCs could be detected in left ventricular samples 30 days after infusion. CONCLUSIONS: Intracoronary infusion of 20 million c-kit positive hCSCs in pigs (equivalent to ~40 million hCSCs in humans) does not cause acute cardiac injury, impairment of cardiac function, or liver and renal injury. These results have immediate translational value and lay the groundwork for using doses of CSCs >1 million in future clinical trials. Further studies are needed to ascertain whether administration of >1 million hCSCs is associated with greater efficacy in patients with ischemic cardiomyopathy.

The NHLBI-sponsored Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR): a new paradigm for rigorous, accurate, and reproducible evaluation of putative infarct-sparing interventions in mice, rabbits, and pigs.

RATIONALE: Despite 4 decades of intense effort and substantial financial investment, the cardioprotection field has failed to deliver a single drug that effectively reduces myocardial infarct size in patients. A major reason is insufficient rigor and reproducibility in preclinical studies. OBJECTIVE: To develop a multicenter, randomized, controlled, clinical trial-like infrastructure to conduct rigorous and reproducible preclinical evaluation of cardioprotective therapies. METHODS AND RESULTS: With support from the National Heart, Lung, and Blood Institute, we established the Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR), based on the principles of randomization, investigator blinding, a priori sample size determination and exclusion criteria, appropriate statistical analyses, and assessment of reproducibility. To validate CAESAR, we tested the ability of ischemic preconditioning to reduce infarct size in 3 species (at 2 sites/species): mice (n=22-25 per group), rabbits (n=11-12 per group), and pigs (n=13 per group). During this validation phase, (1) we established protocols that gave similar results between centers and confirmed that ischemic preconditioning significantly reduced infarct size in all species and (2) we successfully established a multicenter structure to support CAESAR's operations, including 2 surgical centers for each species, a Pathology Core (to assess infarct size), a Biomarker Core (to measure plasma cardiac troponin levels), and a Data Coordinating Center-all with the oversight of an external Protocol Review and Monitoring Committee. CONCLUSIONS: CAESAR is operational, generates reproducible results, can detect cardioprotection, and provides a mechanism for assessing potential infarct-sparing therapies with a level of rigor analogous to multicenter, randomized, controlled clinical trials. This is a revolutionary new approach to cardioprotection. Importantly, we provide state-of-the-art, detailed protocols ("CAESAR protocols") for measuring infarct size in mice, rabbits, and pigs in a manner that is rigorous, accurate, and reproducible.

Gene transfer as a strategy to achieve permanent cardioprotection II: rAAV-mediated gene therapy with heme oxygenase-1 limits infarct size 1 year later without adverse functional consequences.

Extensive evidence indicates that heme oxygenase-1 (HO-1) exerts potent cytoprotective effects in response to stress. Previous studies have shown that gene therapy with HO-1 protects against myocardial ischemia/reperfusion injury for up to 8 weeks after gene transfer. However, the long-term effects of HO-1 gene therapy on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the HO-1 gene (rAAV/HO-1) that enables long-lasting transgene expression. Mice received injections in the anterior LV wall of rAAV/LacZ (LacZ group) or rAAV/HO-1 (HO-1 group); 1 year later, they were subjected to a 30-min coronary occlusion (O) and 4 h of reperfusion (R). Cardiac HO-1 gene expression was confirmed at 1 month and 1 year after gene transfer by immunoblotting and immunohistochemistry analyses. In the HO-1 group, infarct size (% of risk region) was dramatically reduced at 1 year after gene transfer (11.2 ± 2.1%, n = 12, vs. 44.7 ± 3.6%, n = 8, in the LacZ group; P < 0.05). The infarct-sparing effects of HO-1 gene therapy at 1 year were as powerful as those observed 24 h after ischemic PC (six 4-min O/4-min R cycles) (15.0 ± 1.7%, n = 10). There were no appreciable changes in LV fractional shortening, LV ejection fraction, or LV end-diastolic or end-systolic diameter at 1 year after HO-1 gene transfer as compared to the age-matched controls or with the LacZ group. Histology showed no inflammation in the myocardium 1 year after rAAV/HO-1-mediated gene transfer. These results demonstrate, for the first time, that rAAV-mediated HO-1 gene transfer confers long-term (1 year), possibly permanent, cardioprotection without adverse functional consequences, providing proof of principle for the concept of achieving prophylactic cardioprotection (i.e., "immunization against infarction").

Intracoronary administration of cardiac stem cells in mice: a new, improved technique for cell therapy in murine models.

A model of intracoronary stem cell delivery that enables transgenesis/gene targeting would be a powerful tool but is still lacking. To address this gap, we compared intracoronary and intramyocardial delivery of lin(-)/c-kit(+)/GFP(+) cardiac stem cells (CSCs) in a murine model of reperfused myocardial infarction (MI). Lin(-)/c-kit(+)/GFP(+) CSCs were successfully expanded from GFP transgenic hearts and cultured with no detectable phenotypic change for up to ten passages. Intracoronary delivery of CSCs 2 days post-MI resulted in significant alleviation of adverse LV remodeling and dysfunction, which was at least equivalent, if not superior, to that achieved with intramyocardial delivery. Compared with intramyocardial injection, intracoronary infusion was associated with a more homogeneous distribution of CSCs in the infarcted region and a greater increase in viable tissue in this region, suggesting greater formation of new cardiomyocytes. Intracoronary CSC delivery resulted in improved function in the infarcted region, as well as in improved global LV systolic and diastolic function, and in decreased LV dilation and LV expansion index; the magnitude of these effects was similar to that observed after intramyocardial injection. We conclude that, in the murine model of reperfused MI, intracoronary CSC infusion is at least as effective as intramyocardial injection in limiting LV remodeling and improving both regional and global LV function. The intracoronary route appears to be superior in terms of uniformity of cell distribution, myocyte regeneration, and amount of viable tissue in the risk region. To our knowledge, this is the first study to report that intracoronary infusion of stem cells in mice is feasible and effective.

Improved synthesis of O-linked, and first synthesis of S- linked, carbohydrate functionalised N-carboxyanhydrides (glycoNCAs).

An improved method for the synthesis of glycosylated N-carboxyanhydrides, which are monomers for glycopeptide synthesis, is presented.

Etomidate and midazolam for procedural sedation: prospective, randomized trial.

OBJECTIVE: To evaluate whether there is a difference in the time of sedation and time to patient disposition in patients undergoing procedural sedation with etomidate and midazolam. METHODS: Prospective, randomized, double-blind trial comparing etomidate (0.10 mg/kg) and midazolam (0.035 mg/kg) for patients requiring procedural sedation for reduction of joint dislocations or long bone fractures. RESULTS: Forty-five patients were enrolled (24 randomized to etomidate, 21 to midazolam). Groups were similar in demographics and analgesic dosing. Mean time of sedation for etomidate was 15 minutes (SD, 10.97) and for midazolam was 32 minutes (SD, 16.13) (P<.001). Mean time to disposition for etomidate was 121 minutes (SD, 73.28) and for midazolam was 111 minutes (SD, 96.36) (P=.700). The mean quality of sedation for etomidate was 7.91 (SD, 1.53) and for midazolam was 7.48 (SD, 2.89) (P=.570). CONCLUSIONS: The use of etomidate compared with midazolam for procedural sedation provides a significant reduction in recovery time, without a reduction in time to patient disposition, while providing equal sedation quality.

Stimulus characteristics and task category dissociate the anterior and posterior aspects of the novelty P3.

The novelty P3 is an event-related potential component that is most often elicited by environmental sounds within the "novelty oddball" paradigm. Within the context of this paradigm, it is not clear if the novelty P3 can be elicited by deviant stimuli regardless of whether they serve as target or nontarget deviants, or to what extent the physical characteristics of the stimulus contributes to the amplitude of the novelty P3. The current study examines this issue by systematically switching target and nontarget deviants between environmental sounds and tonal stimuli. Participants were 36 young adults. Auditory stimuli were 48 unique tones and 48 unique environmental sounds presented under three experimental conditions. The results showed that target and nontarget deviants elicited novelty P3s with anterior and posterior aspects. The major determinant of the extent of the anterior aspect was the degree of difference between the physical characteristics of the deviant stimuli and the standards. By contrast, the major determinant of the posterior aspect was the task relevance of the deviant stimuli.

Age-related changes in neural trace generation of rule-based auditory features.

Young (M = 22) and elderly volunteers (M = 74) participated in an ERP study that examined rule-based feature ion in the auditory modality. Stimuli were either a frequent ascending tone pair or an infrequent descending tone pair. Tone-pairs were presented under three conditions. Physical feature monaural (1 tone pair), feature monaural (10 tone pairs), and feature binaural (10 tone pairs). Volunteers watched a silent movie during ERP recordings. After completion of the ERP session all volunteers participated in a behavioral discrimination task. MMNs were elicited under all three conditions for the young. For the elderly, MMNs were elicited under both monaural conditions but not under binaural conditions. Behavioral discrimination was high under the physical feature condition but fell to near chance under the two rule-based feature conditions for both age groups. Thus rule-based neural representations were generated for both age groups under monaural conditions, but only for the young under binaural conditions, suggesting that there is an age-related decline in the efficacy of integrating multiple sources into a single auditory stream.