Profiling cells with DELs: Small molecule fingerprinting of cell surfaces.

DNA-encoded small molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, it has been used to identify ligands against targets that are soluble or overexpressed on cell surfaces. Here, we report applying cell-based selection methods to profile surfaces of mouse C2C12 myoblasts and myotube cells in an unbiased, target agnostic manner. A panel of on-DNA compounds were identified and confirmed for cell binding selectivity. We optimized the cell selection protocol and employed a novel data analysis method to identify cell selective ligands against a panel of human B and T lymphocytes. We discuss the generality of using this workflow for DNA encoded small molecule library selection and data analysis against different cell types, and the feasibility of applying this method to profile cell surfaces for biomarker and target identification.

Fractured sternal wire causing a cardiac laceration.

BACKGROUND: Hemopericardium is a serious complication that can occur after cardiac surgery. While most post-operative causes are due to inflammation and bleeding, patients with broken sternal wires and an unstable sternum may develop hemopericardium from penetrating trauma. CASE PRESENTATION: We present the case of a 62-year-old male who underwent triple coronary bypass surgery and presented five months later with sudden anterior chest wall pain. Chest computed tomography revealed hemopericardium with an associated broken sternal wire that had penetrated into the pericardial space. The patient underwent a redo-sternotomy which revealed a 3.5 cm bleeding, jagged right ventricular laceration that correlated to the imaging findings of a fractured sternal wire projecting in the pericardial space. The laceration was repaired using interrupted 4 - 0 polypropylene sutures in horizontal mattress fashion between strips of bovine pericardium. The patient's recovery was uneventful and he was discharged on post-operative day four without complications. CONCLUSION: Patients with broken sternal wires and an unstable sternum require careful evaluation and management as these may have potentially life-threatening complications if left untreated.

An Automated Virtual Reality Training System for Teacher-Student Interaction: A Randomized Controlled Trial.

BACKGROUND: Shortages in qualified supervision and other resources prevent education personnel from rehearsing effective practices. Interactive simulations, although increasingly used in education, frequently require instructor management. Automated simulations rarely engage trainees in skills related to practice (eg, speech). OBJECTIVE: We evaluated the capability of delivering behavioral skills training through an automated virtual reality (VR) simulation using artificial intelligence to improve the implementation of a nondirective mathematical questioning strategy. METHODS: We recruited and randomly assigned 30 college-aged participants to equivalent treatment (ie, lecture, modeling, and VR; 15/30, 50%) and control groups (ie, lecture and modeling only; 15/30, 50%). The participants were blind to treatment conditions. Sessions and assessments were conducted face to face and involved the use of VR for assessment regardless of the condition. Lessons concerned the use of a nondirective mathematical questioning strategy in instances where a simulated student provided correct or incorrect answers to word problems. The measures included observed and automated assessments of participant performance and subjective assessments of participant confidence. The participants completed the pretest, posttest, and maintenance probes each week over the course of 3 weeks. RESULTS: A mixed ANOVA revealed significant main effects of time (F2,27=124.154; P<.001; ηp2=0.816) and treatment (F1,28=19.281; P<.001; ηp2=0.408) as well as an interaction effect (F2,28=8.429; P<.001; ηp2=0.231) for the average percentage of steps in the questioning procedure. Posttest scores for the intervention group (mean 88%, SD 22.62%) exceeded those of the control group (mean 63.33%, SD 22.64%), with t28=3.653, P<.001, and Cohen d=1.334. Maintenance scores indicated a positive effect of the intervention (mean 83.33%, SD 24.40%) relative to the control (mean 54.67%, SD 15.98%), t28=3.807, P<.001, Cohen d=1.39. A Mann-Whitney U test indicated that the treatment groups' self-ratings of confidence (mean 2.41, SD 0.51) were higher than those of the control group (mean 2.04, SD 0.52), U=64, P=.04, r=0.137. CONCLUSIONS: The results demonstrate the potential of artificial intelligence-augmented VR to deliver effective, evidence-based training with limited instructor management. Additional work is needed to demonstrate the cascading effect of training on authentic practice and to encompass a wider range of skills.

Bilateral cardiac sympathetic denervation on an ECMO patient for refractory ventricular arrhythmia: A case report.

Genetic syndrome such as Brugada syndrome can lead to lethal ventricular arrhythmias. Cardiac sympathetic denervation has been shown to be effective in ameliorating refractory ventricular arrhythmias. We present a 33-year-old black female with a medical history of Brugada syndrome with an implantable cardiac defibrillator who presented with refractory ventricular tachycardia leading to cardiogenic shock, requiring extracorporeal membrane oxygenation. The patient subsequently underwent bilateral stellate ganglion sympathetic denervation in the setting of refractory ventricular arrhythmias. We present this case report to showcase that thoracoscopic bilateral cardiac sympathetic denervation can be an effective treatment option for ventricular arrhythmias refractory to medical management.

Exhaled volatile organic compounds and lung microbiome in COPD: a pilot randomised controlled trial.

BACKGROUND: Breath analysis is a burgeoning field, with interest in volatile organic compounds (VOCs) as a noninvasive diagnostic tool or an outcome measure, but no randomised controlled trials (RCTs) have yet evaluated this technology in a clinical trial longitudinally. In a pilot RCT, our exploratory objectives were feasibility of measuring VOCs via multiple techniques, assessing relationships between VOCs and Haemophilus colonisation and whether CXCR2 antagonism with danirixin altered lung microbiome composition in individuals with COPD. METHOD: 43 participants had VOCs and sputum biomarkers evaluated. VOCs and induced sputum were collected after 6 h of fasting at screening and at days 1, 7 and 14. VOCs were analysed via gas chromatography mass spectrometry (GC-MS), field asymmetric ion mobility spectrometry (FAIMS) and eNose. The primary outcome for these analyses was the relationship between VOCs and Haemophilus abundance determined by 16S rRNA sequencing. RESULTS: A joint-effects model demonstrated a modest relationship between four exhaled VOCs and Haemophilus relative abundance (R2=0.55) measured only by GC-MS, but not as measured using gas chromtaography FAIMS or eNose. There was considerable variability in absolute quantities of individual VOCs longitudinally. CONCLUSIONS: VOC measurement in clinical trials to identify subsets of COPD is feasible, but assessment of new VOC technologies must include concurrent GC-MS validation. Further work to standardise collection of VOCs and measuring a background or "housekeeper" VOC is required to understand and normalise individual VOC quantities.

Scaffold-Based Analytics: Enabling Hit-to-Lead Decisions by Visualizing Chemical Series Linked across Large Datasets.

We present a method for visualizing and navigating large screening datasets while also taking into account their activities and properties. Our approach is to annotate the data with all possible scaffolds contained within each molecule. We have developed a Spotfire visualization, coupled to a fuzzy clustering approach based on the scaffold decomposition of the screening deck, used to drive the hit triage process. Progression decisions can be made using aggregate scaffold parameters and data from multiple datasets merged at the scaffold level. This visualization reveals overlaps that help prioritize hits, highlight tractable series, and posit ways to combine aspects of multiple hits. The structure-activity relationship of a large and complex hit is automatically mapped onto all constituent scaffolds making it possible to navigate, via any shared scaffold, to all related hits. This scaffold "walking" helps address bias toward a handful of potent and ligand-efficient molecules at the expense of coverage of chemical space. We consider two scaffold generation methods and explored their similarities and differences both qualitatively and quantitatively. The workflow of a Spotfire visualization used in combination with fuzzy clustering and structure annotation provides an intuitive view of large and diverse screening datasets. This allows teams to effortlessly navigate between structurally related molecules and enriches the population of leads considered and progressed in a manner complementary to established approaches.

Analyzing and comparing complex environmental time series using a cumulative sums approach.

Cumulative sums (Cusums) are a simple, efficient statistical method developed for process control and increasingly used to determine underlying features of time series. Here, two useful applications of Cusums to environmental time series are presented: Cusums in the time domain and plotting Cusum-transformed variables against non-transformed variables to extract meaning in the context of driver-response relationships. These statistical analyses are simple to conduct and provide valuable information about trends, patterns and thresholds of time-series over time and in relation to potential driver variables. In addition, this work investigates the robustness of the Cusum transform to various characteristics of environmental time series that challenge conventional statistical methods. In summary, this work presents: •Cusum methods to derive meaning from complex environmental time series.•Effects of common time series issues on the Cusums method.•Application to real-world datasets.

The benefits of digital drainage system versus traditional drainage system after robotic-assisted pulmonary lobectomy.

BACKGROUND: Postoperative air leaks are the most common complication after a pulmonary resection. There is no data in the literature comparing the traditional and digital chest drainage system after a robotic-assisted pulmonary lobectomy. METHODS: This was a retrospective, correlational study. Medical records from 182 eligible robotic-assisted lobectomy patients were evaluated to determine the association between digital and traditional chest tube drainage systems (CTDS) with postoperative chest tube days, hospital LOS, chest tube reinsertion during hospitalization, and 30-day readmission for pneumothorax. Multiple regression was used to determine the association between CTDS while controlling for confounding variables. RESULTS: No differences were noted between groups for age, gender, BMI, smoking, adhesions or neoadjuvant therapy. Patients with digital drainage systems had significantly shorter chest tube duration than those with traditional drainage systems (2.07 vs. 2.73 days, P=0.003). After controlling for age and BMI, CTDS was not found to be a significant predictor of CT duration. Digital drainage system were also associated with significantly shorter hospital LOS (4.02 vs. 5.06 days, P=0.01) After controlling for age, BMI, and presence of post-op a-fib, use of a digital CTDS was significantly associated with 1 day shorter hospital LOS. Chest tube reinsertion occurred four times more frequently with traditional drainage systems, but the difference did not achieve the level of statistical significance (P=0.059). The frequency of readmission due to pneumothorax was very low (1 patient per group), which prevented comparative statistical analysis. CONCLUSIONS: In the digital drainage system there are shorter chest tube days and hospital length of stay after a robotic-assisted lobectomy. The decision to remove chest tubes in the traditional drainage system is burdened with uncertainty. The digital drainage system reduces intraobserver variability allowing for improved decision making in chest tube removal. Both CT duration and hospital LOS were shorter using unadjusted analyses. Type of CTDS was not significantly associated with CT duration after controlling for age and BMI. However, after controlling for age, BMI, and post-op atrial fibrillation, use of the digital CTDS was associated with a 1 day reduction in hospital LOS.

Screen for modulators of atonal homolog 1 gene expression using notch pathway-relevant gene transcription based cellular assays.

Atonal homolog 1 (Atoh1) is a basic helix-loop-helix 9 (bHLH) transcription factor acting downstream of Notch and is required for the differentiation of sensory hair cells in the inner ear and the specification of secretory cells during the intestinal crypt cell regeneration. Motivated by the observations that the upregulation of Atoh1 gene expression, through genetic manipulation or pharmacological inhibition of Notch signaling (e.g. γ-secretase inhibitors, GSIs), induces ectopic hair cell growth in the cochlea of the inner ear and partially restores hearing after injuries in experimental models, we decided to identify small molecule modulators of the Notch-Atoh1 pathway, which could potentially regenerate hair cells. However, the lack of cellular models of the inner ear has precluded the screening and characterization of such modulators. Here we report using a colon cancer cell line LS-174T, which displays Notch inhibition-dependent Atoh1 expression as a surrogate cellular model to screen for inducers of Atoh1 expression. We designed an Atoh1 promoter-driven luciferase assay to screen a target-annotated library of ~6000 compounds. We further developed a medium throughput, real-time quantitative RT-PCR assay measuring the endogenous Atoh1 gene expression to confirm the hits and eliminate false positives from the reporter-based screen. This strategy allowed us to successfully recover GSIs of known chemotypes. This LS-174T cell-based assay directly measures Atoh1 gene expression induced through Notch-Hes1 inhibition, and therefore offers an opportunity to identify novel cellular modulators along the Notch-Atoh1 pathway.

Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell.

Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROß, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft.

A rare case of sinus of valsalva-right atrial fistula secondary to an abscess perforation from underlying aortic valve endocarditis.

Sinus of Valsalva-right atrial fistulas are abnormal connections between the aorta and the right atrium, and present challenging surgical conditions. An extremely rare etiology of aorto-right atrial fistula is infective endocarditis. This case report presents a 21 year old Caucasian female patient who had native aortic valve Staphylococcus aureus endocarditis complicated by sinus of Valsalva abscess perforation associated with an acute heart block, an aorto-right atrial fistula, severe heart failure, and cardiogenic shock. She underwent emergent aortic valve replacement and complex sinus of Valsalva fistula pericardial patch reconstruction and repair. This case report further explores the advantages and disadvantages of different valves for different patient populations, and evaluates the patient's prosthesis mismatch and effective orifice area.

Changes in sediment bacterial community in response to long-term nutrient enrichment in a subtropical seagrass-dominated estuary.

Florida Bay exhibits a natural gradient of strong P limitation in the east which shifts to weak P or even N limitation at the western boundary. This nutrient gradient greatly affects seagrass abundance and productivity across the bay. We assessed the effects of N and P additions on sediment bacterial community structure in relation to the existing nutrient gradient in Florida Bay. Sediment samples from 24 permanent 0.25 m(2) plots in each of six sites across Florida Bay were fertilized with granular N and P in a factorial design for 26 months. Sediment bacterial community structure was analyzed using PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal RNA (rRNA) genes and a cloning strategy from DGGE bands. The phylogenetic positions of 16S rRNA sequences mostly fell into common members found in marine sediments such as sulfate-reducing Deltaproteobacteria, Gammaproteobacteria, Spirochaetes, and Bacteriodetes. Twenty-eight common DGGE bands were found in all sediment samples; however, some DGGE bands were only found or were better represented in eastern sites. Bacterial community diversity (Shannon-Weiner index) showed similar values throughout all sediment samples. The N treatment had no effect on the bacterial community structures across the bay. Conversely, the addition of P significantly influenced the bacterial community structure at all but the most western site, where P is least limiting due to inputs from the Gulf of Mexico. P additions enhanced DGGE band sequences related to Cytophagales, Ectothiorhodospiraceae, and Desulfobulbaceae, suggesting a shift toward bacterial communities with increased capability to degrade polymeric organic matter. In addition, a band related to Deferribacteres was enhanced in eastern sites. Thus, indigenous environmental conditions were the primary determining factors controlling the bacterial communities, while the addition of P was a secondary determining factor. This P-induced change in community composition tended to be proportional to the amount of P limitation obviated by the nutrient additions.

Biogeochemical classification of South Florida's estuarine and coastal waters.

South Florida's watersheds have endured a century of urban and agricultural development and disruption of their hydrology. Spatial characterization of South Florida's estuarine and coastal waters is important to Everglades' restoration programs. We applied Factor Analysis and Hierarchical Clustering of water quality data in tandem to characterize and spatially subdivide South Florida's coastal and estuarine waters. Segmentation rendered forty-four biogeochemically distinct water bodies whose spatial distribution is closely linked to geomorphology, circulation, benthic community pattern, and to water management. This segmentation has been adopted with minor changes by federal and state environmental agencies to derive numeric nutrient criteria.

The EBM-DPSER conceptual model: integrating ecosystem services into the DPSIR framework.

There is a pressing need to integrate biophysical and human dimensions science to better inform holistic ecosystem management supporting the transition from single species or single-sector management to multi-sector ecosystem-based management. Ecosystem-based management should focus upon ecosystem services, since they reflect societal goals, values, desires, and benefits. The inclusion of ecosystem services into holistic management strategies improves management by better capturing the diversity of positive and negative human-natural interactions and making explicit the benefits to society. To facilitate this inclusion, we propose a conceptual model that merges the broadly applied Driver, Pressure, State, Impact, and Response (DPSIR) conceptual model with ecosystem services yielding a Driver, Pressure, State, Ecosystem service, and Response (EBM-DPSER) conceptual model. The impact module in traditional DPSIR models focuses attention upon negative anthropomorphic impacts on the ecosystem; by replacing impacts with ecosystem services the EBM-DPSER model incorporates not only negative, but also positive changes in the ecosystem. Responses occur as a result of changes in ecosystem services and include inter alia management actions directed at proactively altering human population or individual behavior and infrastructure to meet societal goals. The EBM-DPSER conceptual model was applied to the Florida Keys and Dry Tortugas marine ecosystem as a case study to illustrate how it can inform management decisions. This case study captures our system-level understanding and results in a more holistic representation of ecosystem and human society interactions, thus improving our ability to identify trade-offs. The EBM-DPSER model should be a useful operational tool for implementing EBM, in that it fully integrates our knowledge of all ecosystem components while focusing management attention upon those aspects of the ecosystem most important to human society and does so within a framework already familiar to resource managers.

On the catalytic mechanism of human ATP citrate lyase.

ATP citrate lyase (ACL) catalyzes an ATP-dependent biosynthetic reaction which produces acetyl-coenzyme A and oxaloacetate from citrate and coenzyme A (CoA). Studies were performed with recombinant human ACL to ascertain the nature of the catalytic phosphorylation that initiates the ACL reaction and the identity of the active site residues involved. Inactivation of ACL by treatment with diethylpyrocarbonate suggested the catalytic role of an active site histidine (i.e., His760), which was proposed to form a phosphohistidine species during catalysis. The pH-dependence of the pre-steady-state phosphorylation of ACL with [γ-(33)P]-ATP revealed an ionizable group with a pK(a) value of ~7.5, which must be unprotonated for the catalytic phosphorylation of ACL to occur. Mutagenesis of His760 to an alanine results in inactivation of the biosynthetic reaction of ACL, in good agreement with the involvement of a catalytic histidine. The nature of the formation of the phospho-ACL was further investigated by positional isotope exchange using [γ-(18)O(4)]-ATP. The ß,γ-bridge to nonbridge positional isotope exchange rate of [γ-(18)O(4)]-ATP achieved its maximal rate of 14 s(-1) in the absence of citrate and CoA. This rate decreased to 5 s(-1) when citrate was added, and was found to be 10 s(-1) when both citrate and CoA were present. The rapid positional isotope exchange rates indicated the presence of one or more catalytically relevant, highly reversible phosphorylated intermediates. Steady-state measurements in the absence of citrate and CoA showed that MgADP was produced by both wild type and H760A forms of ACL, with rates at three magnitudes lower than that of k(cat) for the full biosynthetic reaction. The ATPase activity of ACL, along with the small yet significant positional isotope exchange rate observed in H760A mutant ACL (~150 fold less than wild type), collectively suggested the presence of a second, albeit unproductive, phosphoryl transfer in ACL. Mathematical analysis and computational simulation suggested that the desorption of MgADP at a rate of ~7 s(-1) was the rate-limiting step in the biosynthesis of AcCoA and oxaloacetate.

A tale of two subunits: how the neomorphic R132H IDH1 mutation enhances production of αHG.

Heterozygously expressed single-point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2, respectively) render these dimeric enzymes capable of producing the novel metabolite α-hydroxyglutarate (αHG). Accumulation of αHG is used as a biomarker for a number of cancer types, helping to identify tumors with similar IDH mutations. With IDH1, it has been shown that one role of the mutation is to increase the rate of conversion from αKG to αHG. To improve our understanding of the function of this mutation, we have detailed the kinetics of the normal (isocitrate to αKG) and neomorphic (αKG to αHG) reactions, as well as the coupled conversion of isocitrate to αHG. We find that the mutant IDH1 is very efficient in this coupled reaction, with the ability to form αHG from isocitrate and NADP(+). The wild type/wild type IDH1 is also able to catalyze this conversion, though it is much more sensitive to concentrations of isocitrate. This difference in behavior can be attributed to the competitive binding between isocitrate and αKG, which is made more favorable for αKG by the neomorphic mutation at arginine 132. Thus, each partial reaction in the heterodimer is functionally isolated from the other. To test whether there is a cooperative effect resulting from the two subunits being in a dimer, we selectively inactivated each subunit with a secondary mutation in the NADP/H binding site. We observed that the remaining, active subunit was unaffected in its associated activity, reinforcing the notion of each subunit being functionally independent. This was further demonstrated using a monomeric form of IDH from Azotobacter vinelandii, which can be shown to gain the same neomorphic reaction when a homologous mutation is introduced into that protein.

Epiphyte loads on seagrasses and microphytobenthos abundance are not reliable indicators of nutrient availability in oligotrophic coastal ecosystems.

Despite marked gradients in nutrient availability that control the abundance and species composition of seagrasses in south Florida, and the importance of nutrient availability in controlling abundance and composition of epiphytes on seagrasses in other locations, we did not find that epiphyte load on the dominant seagrass, Thalassia testudinum, or that the relative contribution of algal epiphytes to the epiphyte community, was positively correlated with nutrient availability in the water column or the sediment in oligotrophic seagrass beds. Further, the abundance of microphytobenthos, as indicated by Chlorophyll-a concentration in the sediments, was not directly correlated with concentrations of nutrients in the sediments. Our results suggest that epiphyte and microphytobenthos abundance are not unambiguous indicators of nutrient availability in relatively pristine seagrass environments, and therefore would make poor candidates for indicators of the status and trends of seagrass ecosystems in relatively low-nutrient environments like the Florida Keys.