Inserting “OFF-to-ON” BODIPY Tags into Cytokines: A Fluorogenic Interleukin IL-33 for Real-Time Imaging of Immune Cells

The essential functions that cytokine/immune cell interactions play in tissue homeostasis and during disease have prompted the molecular design of targeted fluorophores to monitor their activity in real time. Whereas activatable probes for imaging immune-related enzymes are common, many immunological functions are mediated by binding events between cytokines and their cognate receptors that are hard to monitor by live-cell imaging. A prime example is interleukin-33 (IL-33), a key cytokine in innate and adaptive immunity, whose interaction with the ST2 cell-surface receptor results in downstream signaling and activation of NF-κB and AP-1 pathways. In the present work, we have designed a chemical platform to site-specifically introduce OFF-to-ON BODIPY fluorophores into full cytokine proteins and generate the first native-like fluorescent analogues of IL-33. Among different incorporation strategies, chemical aminoacylation followed by bioorthogonal derivatization led to the best labeling results.Importantly, the BODIPY-labeled IL-33 derivatives -unlike IL-33-GFP constructs- exhibited ST2-specific binding and downstream bioactivity profiles comparable to those of the wild-type interleukin. Real-time fluorescence microscopy assays under no wash conditions confirmed the internalization of IL-33 through ST2 receptors and its intracellular trafficking through the endosomal pathway. We envision that the modularity and versatility of our BODIPY labeling platform will facilitate the synthesis of minimally tagged fluorogenic cytokines as the next generation of imaging reagents for real-time visualization of signaling events in live immune cells

Heliox for croup in children (Review)

Background: Croup is thought to be triggered by a viral infection and is characterised by respiratory distress due to upper airway inflammation and swelling of the subglottic mucosa in children. Mostly it is mild and transient and resolves with supportive care. In moderate to severe cases, treatment with corticosteroids and nebulised epinephrine (adrenaline) is required. Corticosteroids improve symptoms but it takes time for a full effect to be achieved. In the interim, the child is at risk of further deterioration. This may rarely result in respiratory failure necessitating emergency intubation and ventilation. Nebulised epinephrine may result in dose-related adverse effects including tachycardia, arrhythmias and hypertension and its benefit may be short-lived. Helium-oxygen (heliox) inhalation has shown therapeutic benefit in initial treatment of acute respiratory syncytial virus (RSV) bronchiolitis and may prevent morbidity and mortality in ventilated neonates. Heliox has been used during emergency transport of children with severe croup and anecdotal evidence suggests that heliox relieves respiratory distress. Objectives: To examine the effect of heliox on relieving symptoms and signs of croup, as determined by a croup score (a tool for measuring the severity of croup).To examine the effect of croup on rates of admission or intubation (or both), through comparisons of heliox with placebo or any active intervention(s) in children with croup. Search methods: We searched CENTRAL 2013, Issue 10, MEDLINE (1950 to October week 5, 2013), EMBASE (1974 to November 2013), CINAHL (1982 to November 2013), Web of Science (1955 to November 2013) and LILACS (1982 to November 2013). In addition, we searched two clinical trials registries: the World Health Organization International Clinical Trials Registry Platform (WHO ICTRP) and clinicaltrials.gov (searched 12 November 2013). Selection criteria: Randomised controlled trials (RCTs) and quasi-RCTs comparing the effect of helium-oxygen mixtures with placebo or any active intervention(s) in children with croup. Data collection and analysis: Two review authors independently identified and assessed citations for inclusion. A third review author resolved disagreements. We assessed included trials for allocation concealment, blinding of intervention, completeness of outcome data, selective outcome reporting and other potential sources of bias. We reported mean differences for continuous data and odds ratios for dichotomous data. We descriptively reported data not suitable for statistical analysis. Main results: We included three RCTs with a total of 91 participants. One study compared heliox 70%/30% with 30% humidified oxygen administered for 20 minutes in children with mild croup and found no statistically significant differences in the overall change in croup scores between heliox and the comparator. In another study, children with moderate to severe croup were administered intramuscular dexamethasone 0.6 mg/kg and either heliox 70%/30% with one to two doses of nebulised saline, or 100% oxygen with one to two doses of nebulised racaemic epinephrine for three hours. In this study, the heliox group's croup scores improved significantly more at all time points from 90 minutes onwards. However, overall there were no significant differences in croup scores between the groups after four hours using repeated measures analysis. In a third study, children with moderate croup all received one dose of oral dexamethasone 0.3 mg/kg with heliox 70%/30% for 60 minutes in the intervention group and no treatment in the comparator. There was a statistically significant difference in croup scores at 60 minutes in favour of heliox but no significant difference after 120 minutes. It was not possible to pool outcomes because the included studies compared different interventions and reported different outcomes. No adverse events were reported. Authors' conclusions: There is some evidence to suggest a short-term benefit of heliox inhalation in children with moderate to severe croup who have been administered oral or intramuscular dexamethasone. In one study, the benefit appeared to be similar to a combination of 100% oxygen with nebulised epinephrine. In another study there was a slight change in croup scores between heliox and controls, with unclear clinical significance. In another study in mild croup, the benefit of humidified heliox was equivalent to that of 30% humidified oxygen, suggesting that heliox is not indicated in this group of patients provided that 30% oxygen is available. Adequately powered RCTs comparing heliox with standard treatments are needed to further assess the role of heliox in children with moderate to severe croup

Rapid discovery and evolution of nanosensors containing fluorogenic amino acids

Binding-activated optical sensors are powerful tools for imaging, diagnostics, and biomolecular sensing. However, biosensor discovery is slow and requires tedious steps in rational design, screening, and characterization. Here we report on a platform that streamlines biosensor discovery and unlocks directed nanosensor evolution through genetically encodable fluorogenic amino acids (FgAAs). Building on the classical knowledge-based semisynthetic approach, we engineer ~15 kDa nanosensors that recognize specific proteins, peptides, and small molecules with up to 100-fold fluorescence increases and subsecond kinetics, allowing real-time and wash-free target sensing and live-cell bioimaging. An optimized genetic code expansion chemistry with FgAAs further enables rapid (~3 h) ribosomal nanosensor discovery via the cell-free translation of hundreds of candidates in parallel and directed nanosensor evolution with improved variant-specific sensitivities (up to ~250-fold) for SARS-CoV-2 antigens. Altogether, this platform could accelerate the discovery of fluorogenic nanosensors and pave the way to modify proteins with other non-standard functionalities for diverse applications.</p

Raw NMR Data for Template-independent enzymatic synthesis of RNA oligonucleotides

For the characterization of nucleoside triphosphates. The data consists of raw .fid files for 31 phosphorus NMR and 1H NM

Natural Language Processing of Stories

In this thesis, I deal with the task of computationally processing stories with a focus on multidisciplinary ends, specifically in Digital Humanities and Cultural Analytics. In the process, I collect, clean, investigate, and predict from two datasets. The first is a dataset of 2,302 open-source literary works categorized by the time period they are set in. These works were all collected from Project Gutenberg. The classification of the time period in which the work is set was discovered by collecting and inspecting Library of Congress subject classifications, Wikipedia Categories, and literary factsheets from SparkNotes. The second is a dataset of 6,991 open-source literary works categorized by the hierarchical location the work is set in; these labels were constructed from Library of Congress subject classifications and SparkNotes factsheets. These datasets are the first of their kind and can help move forward an understanding of 1) the presentation of settings in stories and 2) the effect the settings have on our understanding of the stories.</p

Natural Language Processing of Stories

Indiana University-Purdue University Indianapolis (IUPUI)In this thesis, I deal with the task of computationally processing stories with a focus on multidisciplinary ends, specifically in Digital Humanities and Cultural Analytics. In the process, I collect, clean, investigate, and predict from two datasets. The first is a dataset of 2,302 open-source literary works categorized by the time period they are set in. These works were all collected from Project Gutenberg. The classification of the time period in which the work is set was discovered by collecting and inspecting Library of Congress subject classifications, Wikipedia Categories, and literary factsheets from SparkNotes. The second is a dataset of 6,991 open-source literary works categorized by the hierarchical location the work is set in; these labels were constructed from Library of Congress subject classifications and SparkNotes factsheets. These datasets are the first of their kind and can help move forward an understanding of 1) the presentation of settings in stories and 2) the effect the settings have on our understanding of the stories

Natural Language Processing of Stories

Indiana University-Purdue University Indianapolis (IUPUI)In this thesis, I deal with the task of computationally processing stories with a focus on multidisciplinary ends, specifically in Digital Humanities and Cultural Analytics. In the process, I collect, clean, investigate, and predict from two datasets. The first is a dataset of 2,302 open-source literary works categorized by the time period they are set in. These works were all collected from Project Gutenberg. The classification of the time period in which the work is set was discovered by collecting and inspecting Library of Congress subject classifications, Wikipedia Categories, and literary factsheets from SparkNotes. The second is a dataset of 6,991 open-source literary works categorized by the hierarchical location the work is set in; these labels were constructed from Library of Congress subject classifications and SparkNotes factsheets. These datasets are the first of their kind and can help move forward an understanding of 1) the presentation of settings in stories and 2) the effect the settings have on our understanding of the stories