Characterizing the Impact of Primer-Template Mismatches on Recombinase Polymerase Amplification.

Recombinase polymerase amplification (RPA) is an isothermal amplification assay that has been ubiquitously utilized in the detection of infectious agents. Like any nucleic acid amplification technology, primer-template complementarity is critical to RPA reaction success. Mismatches arising in the primer-template complex are known to impact reaction kinetics, invalidate downstream analysis, such as nucleic acid quantification, and result in false negatives if used in a diagnostic capacity. Although the impact of specific primer-template mismatches has been well characterized for techniques such as PCR, characterization remains limited for RPA. Through our study, we systematically characterize the impact of mismatches on the RPA reaction, when located in the 3'-anchor region of the primer-template complex. Our investigation identified that the nucleotides involved, as well as position of each mismatch, influence the size of the impact, with terminal cytosine-thymine and guanine-adenine mismatches being the most detrimental. The presence of some mismatch combinations, such as a penultimate cytosine-cytosine and a terminal cytosine-adenine mismatch pairing, led to complete RPA reaction inhibition. Through the successful characterization of 315 mismatch combinations, researchers can optimize their RPA assay accordingly and seek to implement RPA technology for rapid, in-field genotyping.

Thermodynamics of amyloid dissociation provide insights into aggregate stability regimes.

Amyloid aggregates have been hypothesized as a global low free energy state for proteins at finite concentrations. Near its midpoint unfolding temperature, α-chymotrypsinogen A (aCgn) spontaneously forms amyloid polymers, indicating the free energy of aggregates (A) is significantly lower than that for unfolded (U) and native (N) monomers at those particular conditions. The relative thermodynamic stability of A, U, and N states was estimated semi-quantitatively as a function of temperature (T) and [urea] via a combination of calorimetry, urea-assisted unfolding and dissociation, aggregation kinetics, and changes in solvent-exposed surface area, combined with thermodynamic integration and a linear transfer free energy model. The results at first suggest that N is more thermodynamically stable than A at sufficiently low T and [urea], but this may be convoluted with kinetic effects. Interestingly, the kinetic stability of aggregates highlights that the practical measure of stability may be the free energy barrier(s) between A and U, as U serves as a key intermediate between N and A states.

Nucleation, growth, and activation energies for seeded and unseeded aggregation of alpha-chymotrypsinogen A.

The intrinsic time scales for nonnative aggregate nucleation (tau0(n)) and chain growth (tau0(g)) were determined for alpha-chymotrypsinogen A as a function of temperature under acidic conditions where the resulting aggregates do not appreciably condense. Previous results (Andrews and Roberts (2007) Biochemistry 46, 7558) indicated that the product tau0(n)tau0(g) increases with increasing temperature but could not distinguish tau0(n) and tau0(g). Separate experimental values of tau0(n) and tau0(g) are reported here from two approaches based on either (i) combining unseeded monomer loss kinetics with static light scattering of the resulting aggregates or (ii) seeded monomer loss kinetics as a function of number concentration of seed. Values of tau0(n) and tau0(g) from (i) and (ii) agree quantitatively, and indicate that nucleation has a large, negative effective activation energy (ca. -76 kcal/mol) while growth has at most a weak dependence on temperature. The results are consistent with a model in which nucleation requires significant conformational changes within a nonnative oligomer, beyond those for monomer unfolding. The results more generally illustrate the potential utility of approaches (i) and (ii) for quantitatively determining in vitro tau0(n) and tau0(g) values, as well as how the effects of seeding can be predicted purely from unseeded kinetics and static light scattering measurements prior to significant aggregate condensation.

A Lumry-Eyring nucleated polymerization model of protein aggregation kinetics: 1. Aggregation with pre-equilibrated unfolding.

A mathematical model is presented of the kinetics of non-native protein aggregation that combines Lumry-Eyring and nucleated polymerization (LENP) descriptions. The LENP model is solved for cases in which aggregation rates are slow compared to folding-unfolding equilibration and is shown to be a generalization of a number of previously proposed nucleation-and-growth models for non-native and native protein aggregation. The model solutions exhibit a number of qualitative kinetic regimes. Each regime has a characteristic set of experimental signatures that are related to the relative rates of growth and nucleation as well as to the threshold size at which aggregates condense to form higher-order structures or other phases. Approximate model solutions provide practical rate equations that can be regressed against typical experimental kinetic data to obtain mechanistic parameters characterizing the aggregation pathway. In all kinetic regimes, it is found that observed rate coefficients (kobs) or half-lives (t50) obtained from extent-of-reaction measurements are convolutions of more than one stage in the pathway unless purely seeded growth occurs. Despite this convolution, the combination of apparent reaction order (time domain) and the scaling of kobs or t50 with initial protein concentration provides a means to determine a value for the dominant nucleus size in each case. Additional information, such as equilibrium unfolding thermodynamics and the limiting aggregate size distribution, are required to further deconvolute kobs into intrinsic contributions from nucleation, growth, and conformational changes. The model and analysis are expected to be generally applicable to a wide range of proteins and polypeptides that form non-native aggregates.

Non-native aggregation of alpha-chymotrypsinogen occurs through nucleation and growth with competing nucleus sizes and negative activation energies.

The kinetics and structural transitions of non-native aggregation of alpha-chymotrypsinogen (aCgn) were investigated over a wide range of temperature and initial protein concentration at pH 3.5, where high molecular weight aggregates remained soluble throughout the reaction. A comparison of thermodynamic, kinetic, and spectroscopic data shows that aggregation under non-native-favoring conditions proceeds through a molten globule unfolded monomer state, with a nucleation and growth mechanism. Formation of irreversible aggregates and conversion to beta-sheet secondary structures occur simultaneously without detectable intermediates, suggesting that beta-sheet formation may be a commitment step during the nucleation and growth stages. Analysis of the kinetics using a Lumry-Eyring with nucleated polymerization (LENP) model provides the predominant nucleus size and the product of the intrinsic nucleation and intrinsic growth time scales at each state point. We find that the nucleus size depends on both temperature and protein concentration, and in some cases there is competition between two distinct nucleus sizes. The observed rate coefficient (kobs) for aggregation displays a maximum as a function of temperature because of the competition between folding-unfolding thermodynamics and the intrinsic growth and nucleation rates; the latter contribution has a large, negative activation enthalpy that dominates kobs at elevated temperatures. Temperature-jump experiments reveal that aggregates depolymerize at high temperatures, indicating that they are lower in enthalpy than the free monomer. Overall, the results suggest more generally that non-native aggregation may proceed through more than one nucleus size and that intrinsic kinetics of nucleation and growth may have significant entropic barriers.

Molecular and kinetic comparison of the novel extended-spectrum beta-lactamases CTX-M-25 and CTX-M-26.

CTX-M-25 is a novel extended-spectrum beta-lactamase isolated from a single Canadian Escherichia coli isolate. Susceptibility testing demonstrated that this enzyme confers resistance to both cefotaxime and ceftazidime, but the level of resistance was reduced with the addition of beta-lactamase inhibitors. The bla(CTX-M-25) gene was detected on a 111-kb plasmid. It is a member of the CTX-M-8 group and has the closest amino acid identity (99%; three amino acid substitutions) with CTX-M-26. The bla(CTX-M-26) gene was detected on a 100-kb plasmid isolated from a Klebsiella pneumoniae strain from the United Kingdom, and plasmid profiling revealed that it showed some homology to the bla(CTX-M-25)-harboring plasmid. Both CTX-M genes were located downstream of ISEcp1, although the copy upstream of bla(CTX-M-25) was disrupted by IS50-A. Comparative kinetic studies of recombinant CTX-M-25 and CTX-M-26 enzymes showed that CTX-M-25 has a higher level of ceftazidime hydrolysis (kcat values, 33 and 0.005 s(-1) for CTX-M-25 and CTX-M-26, respectively).

The bactericidal activity of gatifloxacin in plasma and urine.

OBJECTIVE: To investigate the bactericidal activity of gatifloxacin in serum and urine against relevant pathogens. METHODS: Serum and urine samples obtained in a single rising dose pharmacokinetic study were investigated for bactericidal activity. The doses employed were placebo, and 200, 400, 600 and 800 mg of gatifloxacin. RESULTS: The titers obtained reflected the dose and susceptibility of the pathogen. In serum a titer of greater-than-or-equal1:8 was observed for Escherichia coli for >24 h after 400 mg; for Staphylococcus aureus and Acinetobacter baumanii the titer was greater-than-or-equal1:8 for between 12--24 h after 600 mg; for Serratia marcescens it was greater-than-or-equal1:8 for >12 h following 800 mg; and for Streptococcus pneumoniae it was greater-than-or-equal1:8 for 8 h following 800 mg. In urine, the titers were greater, and for Escherichia coli, Staphylococcus saprophyticus, Proteus mirabilis and Enterococcus faecalis the titer was >1:8 for 12--24 h after 200 mg and for 24--36 h after a 400-mg dose. Pseudomonas aeruginosa demonstrated a titer of >1:8 for 18--24 h following 600 mg. CONCLUSIONS: These data suggest that gatifloxacin should be efficacious for a wide range of systemic and urinary tract infections.