A map of transcriptional heterogeneity and regulatory variation in human microglia.

Microglia, the tissue-resident macrophages of the central nervous system (CNS), play critical roles in immune defense, development and homeostasis. However, isolating microglia from humans in large numbers is challenging. Here, we profiled gene expression variation in primary human microglia isolated from 141 patients undergoing neurosurgery. Using single-cell and bulk RNA sequencing, we identify how age, sex and clinical pathology influence microglia gene expression and which genetic variants have microglia-specific functions using expression quantitative trait loci (eQTL) mapping. We follow up one of our findings using a human induced pluripotent stem cell-based macrophage model to fine-map a candidate causal variant for Alzheimer's disease at the BIN1 locus. Our study provides a population-scale transcriptional map of a critically important cell for human CNS development and disease

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Recognition Interface of the Thrombin Binding Aptamer Requires Antiparallel Topology of the Quadruplex Core

Recent advances in G-quadruplex (GQ) studies have provided evidence for their important role in key biological processes (replication, transcription, genome stability, and epigenetics). These findings imply highly specific interactions between GQ structures and cellular proteins. The details of the interaction between GQs and cellular proteins remain unknown. It is now accepted that GQ loop elements play a major role in protein recognition. It remains unclear whether and to what extent the GQ core contributes to maintaining the recognition interface. In the current paper, we used the thrombin binding aptamer as a model to study the effect of modification in the quadruplex core on the ability of aptamer to interact with thrombin. We used alpha-2′-deoxyguanosine and 8-bromo-2′-deoxyguanosine to reconfigure the core or to affect syn–anti preferences of selected dG-residues. Our data suggest that core guanines not only support a particular type of GQ architecture, but also set structural parameters that make GQ protein recognition sensitive to quadruplex topology

EXPANDING THE RECOGNITION INTERFACE OF THE THROMBIN BINDING APTAMER HD1 THROUGH MODIFICATION OF RESIDUES T3 AND T12

Post-SELEX modification of DNA aptamers is an established strategy to improve their affinity or inhibitory characteristics. In this study, we examined the possibility of increasing the recognition interface between the thrombin-binding aptamer HD1 (TBA) and thrombin by adding a chemically modified side chain to selected nucleotide residues. A panel of 22 TBA variants with N3-modified residues T3 and T12 was prepared by a two-step modification procedure. Aptamers were characterized by a combination of biophysical and biochemical methods. We identified mutants with enhanced affinity and improved anticoagulant activity. The crystal structures of thrombin complexes with three selected modified variants revealed that the modified pyrimidine base invariably allocates in proximity to thrombin residues Tyr76 and Ile82 due to the directing role of the unmodified TT loop. The modifications induced an increase in the contact areas between thrombin and the modified TBAs. Comparative analysis of the structural, biochemical, and biophysical data suggests that the non-equivalent binding modes of the mutants with thrombin in the T3- and T12-modified series account for the observed systematic differences in their affinity characteristics. In this study, we show that extending the recognition surface between the protein and modified aptamers is a promising approach that may improve characteristics of aptamer ligands

Expanding the recognition interface of the thrombin-binding aptamer HD1 through modification of residues T3 and T12

Post-SELEX modification of DNA aptamers is an established strategy to improve their affinity or inhibitory characteristics. In this study, we examined the possibility of increasing the recognition interface between the thrombin-binding aptamer HD1 (TBA) and thrombin by adding a chemically modified side chain to selected nucleotide residues. A panel of 22 TBA variants with N3-modified residues T3 and T12 was prepared by a two-step modification procedure. Aptamers were characterized by a combination of biophysical and biochemical methods. We identified mutants with enhanced affinity and improved anticoagulant activity. The crystal structures of thrombin complexes with three selected modified variants revealed that the modified pyrimidine base invariably allocates in proximity to thrombin residues Tyr76 and Ile82 due to the directing role of the unmodified TT loop. The modifications induced an increase in the contact areas between thrombin and the modified TBAs. Comparative analysis of the structural, biochemical, and biophysical data suggests that the non-equivalent binding modes of the mutants with thrombin in the T3- and T12-modified series account for the observed systematic differences in their affinity characteristics. In this study, we show that extending the recognition surface between the protein and modified aptamers is a promising approach that may improve characteristics of aptamer ligands

Oligodeoxynucleotides Containing N1-Methyl-2′-Deoxyadenosine and N6-Methyl-2′-Deoxyadenosine

This unit describes a simple and efficient synthesis of the phosphoramidite derivative of N(1)-methyl-2'-deoxyadenosine from 2'-deoxyadenosine. The synthesis starts with the monomethoxytritylation of 2'-deoxyadenosine followed by methylation of 5'-O-protected nucleoside at N-1. Subsequent N-chloroacetylation leads to N(6)-chloroacetyl-N(1)-methyl-5'-O-(p-anisyldiphenylmethyl)-2'-deoxyadenosine, which is finally converted to its 3' phosphoramidite derivative. This phosphoramidite is used to incorporate N(1)-methyl-2'-deoxyadenosine into synthetic oligonucleotides. N-Chloroacetyl protection and controlled anhydrous deprotection conditions are used to avoid the Dimroth rearrangement.status: publishe

Oligodeoxynucleotides containing 2'-deoxy-1-methyladenosine and Dimroth rearrangement

2’-Deoxy-1-methyladenosine was incorporated into synthetic oligonucleotides by phosphoramidite chemistry. Chloroacetyl protecting group and controlled anhydrous deprotection conditions were used to avoid Dimroth rearrangement. Hybridization studies of intramolecular duplexes showed that introduction of a modified residue into the loop region of the oligonucleotide hairpin increases the melting temperature. It was shown that modified oligonucleotides may be easily transformed into oligonucleotides containing 2’-deoxy-N6-methyladenosine.status: publishe