KPC-39-mediated resistance to Ceftazidime-Avibactam in a Klebsiella pneumoniae ST307 clinical isolate

Resistance to ceftazidime–avibactam (CAZ-AVI) combination is being increasingly reported. Here, we report a CAZ-AVI resistant Klebsiella pneumoniae belonging to the high-risk ST307 clone and producing KPC-39, a single amino-acid variant of KPC-3 (A172T). Cloning experiments, steady state kinetic parameters and molecular dynamics simulations revealed a loss of carbapenemase activity and an increased affinity for ceftazidime. KPC-39 was identified in a patient without prior exposure to CAZ-AVI, suggesting silent dissemination in European healthcare settings.

Determination of Kinetic Properties in Unimolecular Dissociation of Complex Systems from Graph-Theory Based Analysis of an Ensemble of Reactive Trajectories.

<div>We describe and apply a general approach based on graph-theory to obtain kinetic and structural properties from direct dynamics simulations. In particular, we focus on the unimolecular fragmentation of complex systems in which, prior to dissociation, different events can take place, and notably isomerizations and formation of ion-molecule complex.</div><div>3-state and 4-state kinetic models are thus obtained and rate constants for global or specific pathways are obtained from direct counting and flux calculation, both being in agreement.<br />Finally, we show how a theoretical mass spectrum can also be obtained automatically.<br /></div>

Detection and characterization of VIM-52, a new variant of VIM-1 from Klebsiella pneumoniae clinical isolate

Over the last two decades, antimicrobial resistance has become a global health problem. In Gram-negative bacteria, metallo-β-lactamases (MBLs), which inactivate virtually all β-lactams, increasingly contribute to this phenomenon. The aim of this study is to characterize VIM-52, a His224Arg variant of VIM-1, identified in a Klebsiella pneumoniae clinical isolate. VIM-52 conferred lower MICs to cefepime and ceftazidime as compared to VIM-1. These results were confirmed by steady state kinetic measurements, where VIM-52 yielded a lower activity towards ceftazidime and cefepime but not against carbapenems. Residue 224 is part of the L10 loop (residues 221-241), which borders the active site. As Arg 224 and Ser 228 are both playing an important and interrelated role in enzymatic activity, stability and substrate specificity for the MBLs, targeted mutagenesis at both positions were performed and further confirmed their crucial role for substrate specificity.

Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity

The specificity of CRISPR/Cas9 genome editing is largely determined by the sequences of guide RNA (gRNA) and the targeted DNA, yet the sequence-dependent rules underlying off-target effects are not fully understood. Here we systematically investigated the sequence determinants governing CRISPR/Cas9 specificity by measuring the off-on ratios of 1,902 gRNAs on 13,314 target sequences using an improved synthetic system with dual-target design. Our study revealed a comprehensive set of rules including 3 factors in CRISPR/Cas9 off-targeting: 1) the nucleotide context and position of a single mismatch; 2) an epistasis-like combinatorial effect of multiple mismatches; and 3) a guide-intrinsic mismatch tolerance (GMT) independent of the mismatch context. Notably, the combinatorial effect and GMT are associated with the free-energy landscape in R-loop formation and are explainable by a multi-state kinetic model. Based on these rules, we developed a model-based off-target prediction tool (MOFF), which showed superior performance compared to the existing methods.

Steady-state kinetic analysis of mitochondrial respiratory enzymes from bovine heart mitochondria

Mitochondria is a decisive organelle of cells that produces adenosine triphosphate (ATP) by the process of oxidative phosphorylation of the Krebs cycle and the electron transport chain. The electron transport chain system of mitochondria embodies multiple enzyme supercomplexes including complex I to V which located in the inner membrane. Although the simple enzyme activity of some as-isolated complex has been studied so far, the steady-state kinetic analysis of each complex within the form of mitochondrial supercomplex has not been studied in depth. To this end, kinetic parameters of mitochondrial complex I–IV were determined using steady-kinetic analysis using corresponding substrates of them. Catalytic activity and binding affinity between substrates and enzymes were obtained by fitting the data to the Michaelis–Menten equation. Acquired kinetic parameters represented distinctive values depending on the complexes that can be interpreted by the characteristics of the enzymes including the distinction of substrates or the ratio of the enzyme itself under the supercomplex form. The indirect kcat of the mitochondrial enzymes were varied from 0.0609 to 0.334 s−1 in order of complex III, II, I, and IV and Km of substrates were also diverse from 5.1 μM to 12.14 mM. This is the first attempt to get exact kinetic values that should provide profound information to evaluate the mitochondrial function practically in advance.

Determination of Kinetic Properties in Unimolecular Dissociation of Complex Systems from Graph-Theory Based Analysis of an Ensemble of Reactive Trajectories.

<div>We describe and apply a general approach based on graph-theory to obtain kinetic and structural properties from direct dynamics simulations. In particular, we focus on the unimolecular fragmentation of complex systems in which, prior to dissociation, different events can take place, and notably isomerizations and formation of ion-molecule complex.</div><div>3-state and 4-state kinetic models are thus obtained and rate constants for global or specific pathways are obtained from direct counting and flux calculation, both being in agreement.<br>Finally, we show how a theoretical mass spectrum can also be obtained automatically.<br></div>

Determination of Kinetic Properties in Unimolecular Dissociation of Complex Systems from Graph-Theory Based Analysis of an Ensemble of Reactive Trajectories.

<div>We describe and apply a general approach based on graph-theory to obtain kinetic and structural properties from direct dynamics simulations. In particular, we focus on the unimolecular fragmentation of complex systems in which, prior to dissociation, different events can take place, and notably isomerizations and formation of ion-molecule complex.</div><div>3-state and 4-state kinetic models are thus obtained and rate constants for global or specific pathways are obtained from direct counting and flux calculation, both being in agreement.<br>Finally, we show how a theoretical mass spectrum can also be obtained automatically.<br></div>