The NIH-led research response to COVID-19.

Investment, collaboration, and coordination have been key.

The National COVID Cohort Collaborative (N3C): Rationale, design, infrastructure, and deployment.

Objective: Coronavirus disease 2019 (COVID-19) poses societal challenges that require expeditious data and knowledge sharing. Though organizational clinical data are abundant, these are largely inaccessible to outside researchers. Statistical, machine learning, and causal analyses are most successful with large-scale data beyond what is available in any given organization.

Accelerating global innovation to address antibacterial resistance: introducing CARB-X.

A global response to the chronic shortfall in antibiotic innovation is urgently needed to combat antimicrobial resistance. Here, we introduce CARB-X, a new global public-private partnership that will invest more than US$350 million in the next 5 years to accelerate the progression of a diverse portfolio of innovative antibacterial products into clinical trials.

Biocontainment laboratory risk assessment: perspectives and considerations.

The ability to respond to public health emergencies involving infectious diseases as well as our ability to adequately prepare for as yet unknown or unrecognized emerging infectious diseases requires suitable facilities within which scientific investigations can take place. To ensure the safe conduct of such investigations so that laboratory workers and the general public are protected from potential consequences of accidental or intentional release of high consequence pathogens, special containment facilities have been designed and constructed. Evaluation of the adequacy of containment for these types of investigations requires a risk assessment (RA) as part of the overall construction project for these types of laboratories.

High priority preparedness research and its support.

The US Federal Government has considerable interest in supporting research into preparedness. Because of the diverse nature of possible threats and the responsibilities of different agencies, a number of different programs have been developed. Perspectives from representatives from 3 of the leading agencies; the Department of Homeland Security, the Centers from Disease Control and Prevention, and the National Institutes of Health, are described herein.

Catalytic properties of mutant 23 S ribosomes resistant to oxazolidinones.

Kinetic analysis of ribosomal peptidyltransferase activity in a methanolic puromycin reaction with wild type and drug-resistant 23 S RNA mutants was used to probe the structural basis of catalysis and mechanism of resistance to antibiotics. 23 S RNA mutants G2032A and G2447A are resistant to oxazolidinones both in vitro and in vivo with the latter displaying a 5-fold increase in the value of Km for initiator tRNA and a 100-fold decrease in Vmax in puromycin reaction. Comparison of the Ki values for oxazolidinones, chloramphenicol, and sparsomycin revealed partial cross-resistance between oxazolidinones and chloramphenicol; no cross-resistance was observed with sparsomycin, a known inhibitor of the peptidyltransferase A-site.

Regulated expression of the Escherichia coli lepB gene as a tool for cellular testing of antimicrobial compounds that inhibit signal peptidase I in vitro.

Escherichia coli under-expressing lepB was utilized to test cellular inhibition of signal peptidase I (SPase). For the construction of a lepB regulatable strain, the E. coli lepB gene was cloned into pBAD, with expression dependent on L-arabinose.

A multitarget assay for inhibitors of membrane-associated steps of peptidoglycan biosynthesis.

Peptidoglycan synthesis begins in the cytoplasm with the condensation of UDP-N-acetyl glucosamine (UDP-GlcNAc) and phosphoenolpyruvate catalyzed by UDP-N-acetylglucosamine enolpyruvoyl transferase. UDP-GlcNAc is also utilized as substrate for the glycosyltransferase MurG, a membrane-bound enzyme that catalyzes the production of lipid II. Membranes from Escherichia coli cells overproducing MurG support peptidoglycan formation at a rate approximately fivefold faster than membranes containing wild-type levels of MurG.

Homogenous assays for Escherichia coli DnaB-stimulated DnaG primase and DnaB helicase and their use in screening for chemical inhibitors.

Escherichia coli DnaG primase is a single-stranded DNA-dependent RNA polymerase. Primase catalyzes the synthesis of a short RNA primer to initiate DNA replication at the origin and to initiate Okazaki fragment synthesis for synthesis of the lagging strand. Primase activity is greatly stimulated through its interaction with DnaB helicase.

PolC-type polymerase III of Streptococcus pyogenes and its use in screening for chemical inhibitors.

The polC gene from Streptococcus pyogenes (S. pyogenes, strain SF370) has been cloned and expressed in Escherichia coli (E. coli) as a fusion protein containing an N-terminal histidine tag.

Development of a whole-cell assay for peptidoglycan biosynthesis inhibitors.

Osmotically stabilized Escherichia coli cells subjected to freezing and thawing were utilized as the source of enzymes for a peptidoglycan pathway assay that can be used to simultaneously test all targets of the committed steps of cell wall biosynthesis. The use of (14)C-labeled UDP-N-acetylglucosamine (UDP-GlcNAc) as a substrate allows the direct detection of cross-linked peptidoglycan formed. The assay was validated with known antibiotics.