High-throughput Kinetics using capillary Electrophoresis and Robotics (HiKER) platform used to study T7, T3, and Sp6 RNA polymerase misincorporation.

T7 RNA Polymerase (RNAP) is a widely used enzyme with recent applications in the production of RNA vaccines. For over 50 years denaturing sequencing gels have been used as key analysis tools for probing the nucleotide addition mechanisms of T7 RNAP and other polymerases. However, sequencing gels are low-throughput limiting their utility for comprehensive enzyme analysis.

Virus-encoded glycosyltransferases hypermodify DNA with diverse glycans.

Enzymatic modification of DNA nucleobases can coordinate gene expression, nuclease protection, or mutagenesis. We recently discovered a clade of phage-specific cytosine methyltransferase (MT) and 5-methylpyrimidine dioxygenase (5mYOX) enzymes that produce 5-hydroxymethylcytosine (5hmC) as a precursor for enzymatic hypermodifications on viral genomes. Here, we identify phage MT- and 5mYOX-associated glycosyltransferases (GTs) that catalyze linkage of diverse sugars to 5hmC nucleobase substrates.

Impact of a clinical decision support system on identifying drug-related problems and making recommendations to providers during community pharmacist-led medication reviews in Ontario, Canada: A pilot study.

Objective: To evaluate the impact of a clinical decision support system (CDSS) to identify drug-related problems (DRPs) during community pharmacist medication reviews.

Design: Pilot 3-phase (group), open-label study.

Setting And Participants: Two community pharmacies in Sarnia, Ontario, with pharmacists providing medication reviews to patients.

Sialic Acid-Siglec-E Interactions Regulate the Response of Neonatal Macrophages to Group B Streptococcus.

The mammalian Siglec receptor sialoadhesin (Siglec1, CD169) confers innate immunity against the encapsulated pathogen group B Streptococcus (GBS). Newborn lung macrophages have lower expression levels of sialoadhesin at birth compared with the postnatal period, increasing their susceptibility to GBS infection. In this study, we investigate the mechanisms regulating sialoadhesin expression in the newborn mouse lung.

Highly Parallelized Construction of DNA from Low-Cost Oligonucleotide Mixtures Using Data-Optimized Assembly Design and Golden Gate.

Commercially synthesized genes are typically made using variations of homology-based cloning techniques, including polymerase cycling assembly from chemically synthesized microarray-derived oligonucleotides. Here, we apply Data-optimized Assembly Design (DAD) to the synthesis of hundreds of codon-optimized genes in both constitutive and inducible vectors using Golden Gate Assembly. Starting from oligonucleotide pools, we synthesize genes in three simple steps: (1) amplification of parts belonging to individual assemblies in parallel from a single pool; (2) Golden Gate Assembly of parts for each construct; and (3) transformation.

High-Complexity One-Pot Golden Gate Assembly.

Golden Gate Assembly is a flexible method of DNA assembly and cloning that permits the joining of multiple fragments in a single reaction through predefined connections. The method depends on cutting DNA using a Type IIS restriction enzyme, which cuts outside its recognition site and therefore can generate overhangs of any sequence while separating the recognition site from the generated fragment. By choosing compatible fusion sites, Golden Gate permits the joining of multiple DNA fragments in a defined order in a single reaction.

The Lung Microenvironment Instructs Gene Transcription in Neonatal and Adult Alveolar Macrophages.

Immaturity of alveolar macrophages (AMs) around birth contributes to the susceptibility of newborns to lung disease. However, the molecular features differentiating neonatal and mature, adult AMs are poorly understood. In this study, we identify the unique transcriptomes and enhancer landscapes of neonatal and adult AMs in mice.

Immunocompromised Cas9 transgenic mice for rapid assessment of host factors involved in highly pathogenic virus infection.

Targeting host factors for anti-viral development offers several potential advantages over traditional countermeasures that include broad-spectrum activity and prevention of resistance. Characterization of host factors in animal models provides strong evidence of their involvement in disease pathogenesis, but the feasibility of performing high-throughput analyses on lists of genes is problematic. To begin addressing the challenges of screening candidate host factors , we combined advances in CRISPR-Cas9 genome editing with an immunocompromised mouse model used to study highly pathogenic viruses.

Phage-encoded ten-eleven translocation dioxygenase (TET) is active in C5-cytosine hypermodification in DNA.

TET/JBP (ten-eleven translocation/base J binding protein) enzymes are iron(II)- and 2-oxo-glutarate-dependent dioxygenases that are found in all kingdoms of life and oxidize 5-methylpyrimidines on the polynucleotide level. Despite their prevalence, few examples have been biochemically characterized. Among those studied are the metazoan TET enzymes that oxidize 5-methylcytosine in DNA to hydroxy, formyl, and carboxy forms and the euglenozoa JBP dioxygenases that oxidize thymine in the first step of base J biosynthesis.

Developmental Immaturity of Siglec Receptor Expression on Neonatal Alveolar Macrophages Predisposes to Severe Group B Streptococcal Infection.

Streptococcus agalactiae (Group B Streptococcus, GBS) is the most common neonatal pathogen. However, the cellular and molecular mechanisms for neonatal susceptibility to GBS pneumonia and sepsis are incompletely understood. Here we optimized a mouse model of GBS pneumonia to test the role of alveolar macrophage (ΑΜΦ) maturation in host vulnerability to disease.

Differential Immune Activation in Fetal Macrophage Populations.

Distinct macrophage subsets populate the developing embryo and fetus in distinct waves. However little is known about the functional differences between in utero macrophage populations or how they might contribute to fetal and neonatal immunity. Here we tested the innate immune response of mouse macrophages derived from the embryonic yolk sac and from fetal liver.

Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production.

Isoprenoids are a large class of natural products with wide-ranging applications. Synthetic biology approaches to the manufacture of isoprenoids and their new-to-nature derivatives are limited due to the provision in nature of just two hemiterpene building blocks for isoprenoid biosynthesis. To address this limitation, artificial chemo-enzymatic pathways such as the alcohol-dependent hemiterpene (ADH) pathway serve to leverage consecutive kinases to convert exogenous alcohols into pyrophosphates that could be coupled to downstream isoprenoid biosynthesis.

An Artificial Pathway for Isoprenoid Biosynthesis Decoupled from Native Hemiterpene Metabolism.

Isoprenoids are constructed in nature using hemiterpene building blocks that are biosynthesized from lengthy enzymatic pathways with little opportunity to deploy precursor-directed biosynthesis. Here, an artificial alcohol-dependent hemiterpene biosynthetic pathway was designed and coupled to several isoprenoid biosynthetic systems, affording lycopene and a prenylated tryptophan in robust yields. This approach affords a potential route to diverse non-natural hemiterpenes and by extension isoprenoids modified with non-natural chemical functionality.

Approaches and Recent Developments for the Commercial Production of Semi-synthetic Artemisinin.

The antimalarial drug artemisinin is a natural product produced by the plant . Extracts of have been used in Chinese herbal medicine for over two millennia. Following the re-discovery of extract as an effective antimalarial, and the isolation and structural elucidation of artemisinin as the active agent, it was recommended as the first-line treatment for uncomplicated malaria in combination with another effective antimalarial drug (Artemisinin Combination Therapy) by the World Health Organization (WHO) in 2002.

Virulence Role of the GlcNAc Side Chain of the Lancefield Cell Wall Carbohydrate Antigen in Non-M1-Serotype Group A .

Classification of streptococci is based upon expression of unique cell wall carbohydrate antigens. All serotypes of group A (GAS; ), a leading cause of infection-related mortality worldwide, express the group A carbohydrate (GAC). GAC, the classical Lancefield antigen, is comprised of a polyrhamnose backbone with -acetylglucosamine (GlcNAc) side chains.

Direct analysis of terpenes from biological buffer systems using SESI and IR-MALDESI.

Terpenes are the largest class of natural products with a wide range of applications including use as pharmaceuticals, fragrances, flavorings, and agricultural products. Terpenes are biosynthesized by the condensation of a variable number of isoprene units resulting in linear polyisoprene diphosphate units, which can then be cyclized by terpene synthases into a range of complex structures. While these cyclic structures have immense diversity and potential in different applications, their direct analysis in biological buffer systems requires intensive sample preparation steps such as salt cleanup, extraction with organic solvents, and chromatographic separations.

Leukotriene C4 Potentiates IL-33-Induced Group 2 Innate Lymphoid Cell Activation and Lung Inflammation.

Asthma is a complex disease that is promoted by dysregulated immunity and the presence of many cytokine and lipid mediators. Despite this, there is a paucity of data demonstrating the combined effects of multiple mediators in asthma pathogenesis. Group 2 innate lymphoid cells (ILC2s) have recently been shown to play important roles in the initiation of allergic inflammation; however, it is unclear whether lipid mediators, such as cysteinyl leukotrienes (CysLTs), which are present in asthma, could further amplify the effects of IL-33 on ILC2 activation and lung inflammation.

Cyclic stretch of embryonic cardiomyocytes increases proliferation, growth, and expression while repressing Tgf-β signaling.

Perturbed biomechanical stimuli are thought to be critical for the pathogenesis of a number of congenital heart defects, including Hypoplastic Left Heart Syndrome (HLHS). While embryonic cardiomyocytes experience biomechanical stretch every heart beat, their molecular responses to biomechanical stimuli during heart development are poorly understood. We hypothesized that biomechanical stimuli activate specific signaling pathways that impact proliferation, gene expression and myocyte contraction.

Increased ILC2s in the eosinophilic nasal polyp endotype are associated with corticosteroid responsiveness.

Group 2 innate lymphoid cells (ILC2s) have recently been identified in human nasal polyps, but whether numbers of ILC2s differ by polyp endotype or are influenced by corticosteroid use is unknown. Here, we show that eosinophilic nasal polyps contained double the number of ILC2s vs. non-eosinophilic polyps.

Type 2 Innate Lymphoid Cells in Allergic Disease.

Type II innate lymphoid cells (ILC2) are a novel population of lineage-negative cells that produce high levels of Th2 cytokines IL-5 and IL-13. ILC2 are found in human respiratory and gastrointestinal tissue as well as in skin. Studies from mouse models of asthma and atopic dermatitis suggest a role for ILC2 in promoting allergic inflammation.

Allergen challenge in allergic rhinitis rapidly induces increased peripheral blood type 2 innate lymphoid cells that express CD84.

Type 2 innate lymphoid cells (ILC2) produce high levels of Th2 cytokines. Our study demonstrates that cat allergen challenge in allergic rhinitis subjects rapidly induces increased peripheral blood ILC2.

Innate type 2 response to Alternaria extract enhances ryegrass-induced lung inflammation.

Background: Exposure to the fungal allergen Alternaria alternata as well as ryegrass pollen has been implicated in severe asthma symptoms during thunderstorms. We have previously shown that Alternaria extract induces innate type 2 lung inflammation in mice. We hypothesized that the innate eosinophilic response to Alternaria extract may enhance lung inflammation induced by ryegrass.

Impaired induction of allergic lung inflammation by Alternaria alternata mutant MAPK homologue Fus3.

The fungal allergen Alternaria alternata is associated with development of asthma, though the mechanisms underlying the allergenicity of Alternaria are largely unknown. The aim of this study was to identify whether the MAP kinase homologue Fus3 of Alternaria contributed to allergic airway responses. Wild-type (WT) and Fus3 deficient Alternaria extracts were given intranasal to mice.