Population Pharmacokinetics and Dosing Simulation of Vancomycin Administered by Continuous Injection in Critically Ill Patient.

Background: Vancomycin is widely used for empirical antimicrobial therapy in critically ill patients with sepsis. Continuous infusion (CI) may provide more stable exposure than intermittent infusion, but optimal dosing remains challenging. The aims of this study were to perform population pharmacokinetic (PK) analysis of vancomycin administered by CI in intensive care unit (ICU) patients to identify optimal dosages. Methods: Patients who received vancomycin by CI with at least one measured concentration in our center over 16 months were included, including those under continuous renal replacement therapy (CRRT). Population PK was conducted and external validation of the final model was performed in a dataset from another center. Simulations were conducted with the final model to identify the optimal loading and maintenance doses for various stages of estimated creatinine clearance (CRCL) and in patients on CRRT. Target exposure was defined as daily AUC of 400-600 mg·h/L on the second day of therapy (AUC24-48 h). Results: A two-compartment model best described the data. Central volume of distribution was allometrically scaled to ideal body weight (IBW), whereas vancomycin clearance was influenced by CRRT and CRCL. Simulations performed with the final model suggested a loading dose of 27.5 mg/kg of IBW. The maintenance dose ranged from 17.5 to 30 mg/kg of IBW, depending on renal function. Overall, simulation showed that 55.8% (95% CI; 47-64%) of patients would achieve the target AUC with suggested dosages. Discussion: A PK model has been validated for vancomycin administered by CI in ICU patients, including patients under CRRT. Our model-informed precision dosing approach may help for early optimization of vancomycin exposure in such patients.

Limiting homologous recombination at stalled replication forks is essential for cell viability: DNA2 to the rescue.

The disease-associated nuclease-helicase DNA2 has been implicated in DNA end-resection during DNA double-strand break repair, Okazaki fragment processing, and the recovery of stalled DNA replication forks (RFs). Its role in Okazaki fragment processing has been proposed to explain why DNA2 is indispensable for cell survival across organisms. Unexpectedly, we found that DNA2 has an essential role in suppressing homologous recombination (HR)-dependent replication restart at stalled RFs. In the absence of DNA2-mediated RF recovery, excessive HR-restart of stalled RFs results in toxic levels of abortive recombination intermediates that lead to DNA damage-checkpoint activation and terminal cell-cycle arrest. While HR proteins protect and restart stalled RFs to promote faithful genome replication, these findings show how HR-dependent replication restart is actively constrained by DNA2 to ensure cell survival. These new insights disambiguate the effects of DNA2 dysfunction on cell survival, and provide a framework to rationalize the association of DNA2 with cancer and the primordial dwarfism disorder Seckel syndrome based on its role in RF recovery.

Disease-associated DNA2 nuclease-helicase protects cells from lethal chromosome under-replication.

DNA2 is an essential nuclease-helicase implicated in DNA repair, lagging-strand DNA synthesis, and the recovery of stalled DNA replication forks (RFs). In Saccharomyces cerevisiae, dna2Δ inviability is reversed by deletion of the conserved helicase PIF1 and/or DNA damage checkpoint-mediator RAD9. It has been suggested that Pif1 drives the formation of long 5'-flaps during Okazaki fragment maturation, and that the essential function of Dna2 is to remove these intermediates. In the absence of Dna2, 5'-flaps are thought to accumulate on the lagging strand, resulting in DNA damage-checkpoint arrest and cell death. In line with Dna2's role in RF recovery, we find that the loss of Dna2 results in severe chromosome under-replication downstream of endogenous and exogenous RF-stalling. Importantly, unfaithful chromosome replication in Dna2-mutant cells is exacerbated by Pif1, which triggers the DNA damage checkpoint along a pathway involving Pif1's ability to promote homologous recombination-coupled replication. We propose that Dna2 fulfils its essential function by promoting RF recovery, facilitating replication completion while suppressing excessive RF restart by recombination-dependent replication (RDR) and checkpoint activation. The critical nature of Dna2's role in controlling the fate of stalled RFs provides a framework to rationalize the involvement of DNA2 in Seckel syndrome and cancer.

Rif1 S-acylation mediates DNA double-strand break repair at the inner nuclear membrane.

Rif1 is involved in telomere homeostasis, DNA replication timing, and DNA double-strand break (DSB) repair pathway choice from yeast to human. The molecular mechanisms that enable Rif1 to fulfill its diverse roles remain to be determined. Here, we demonstrate that Rif1 is S-acylated within its conserved N-terminal domain at cysteine residues C466 and C473 by the DHHC family palmitoyl acyltransferase Pfa4. Rif1 S-acylation facilitates the accumulation of Rif1 at DSBs, the attenuation of DNA end-resection, and DSB repair by non-homologous end-joining (NHEJ). These findings identify S-acylation as a posttranslational modification regulating DNA repair. S-acylated Rif1 mounts a localized DNA-damage response proximal to the inner nuclear membrane, revealing a mechanism of compartmentalized DSB repair pathway choice by sequestration of a fatty acylated repair factor at the inner nuclear membrane.

Prediction of docetaxel toxicity in older cancer patients: a Bayesian network approach.

Chemotherapy is an essential therapy in the fight against cancer. Polypathology and polymedication are often encountered in elderly patients, making this population especially at risk for adverse drug reactions, and particularly with cytotoxic drugs. The objective of this study was to build a model to predict high-grade toxicity in elderly patients treated with docetaxel. Data from the trial TAX-108 have been used to create the model. The variable to predict was the occurrence of grade 3 or 4 toxicity. The explanatory variables entered in the model were anthropometric and biological characteristics of patients at inclusion; fragility criteria (SMAF, CIRS-G, performance status); location of the primary tumor; chemotherapy history, radiotherapy or surgery; weekly dose of docetaxel, cumulative dose administered. A Bayesian network model was developed using a global search procedure and an Expectation-Maximization algorithm. A 10-fold cross-validation was performed. A toxicity of grade 3 or higher was observed in 54% of patients. The variables providing the most information were the primary site (19.4%), the dose per course (17.5%), and albuminemia (13.1%). The area under the curve of the model obtained after cross-validation was 74 ± 1.4%. The model built allows classifying correctly 71.21 ± 0.9% of patients in our sample in the cross-validation procedure. The sensitivity and specificity of the model were 75 and 67%, respectively, and the positive and negative predictive values were 73 and 69%. The encouraging results from this first study show that Bayesian networks could help assess the benefit-risk ratio of chemotherapy in elderly patients.

Structure-Specific Endonucleases and the Resolution of Chromosome Underreplication.

Complete genome duplication in every cell cycle is fundamental for genome stability and cell survival. However, chromosome replication is frequently challenged by obstacles that impede DNA replication fork (RF) progression, which subsequently causes replication stress (RS). Cells have evolved pathways of RF protection and restart that mitigate the consequences of RS and promote the completion of DNA synthesis prior to mitotic chromosome segregation. If there is entry into mitosis with underreplicated chromosomes, this results in sister-chromatid entanglements, chromosome breakage and rearrangements and aneuploidy in daughter cells. Here, we focus on the resolution of persistent replication intermediates by the structure-specific endonucleases (SSEs) MUS81, SLX1-SLX4 and GEN1. Their actions and a recently discovered pathway of mitotic DNA repair synthesis have emerged as important facilitators of replication completion and sister chromatid detachment in mitosis. As RS is induced by oncogene activation and is a common feature of cancer cells, any advances in our understanding of the molecular mechanisms related to chromosome underreplication have important biomedical implications.

Time in Therapeutic Range of Oral Vitamin K Antagonists in Hospitalized Elderly Patients.

BACKGROUND: Oral vitamin K antagonists (VKAs) are commonly used in older adults. To ensure the efficiency and safety of these drugs, the international normalized ratio (INR) must be monitored. The time in therapeutic range (TTR) is an internationally recommended assessment of the anticoagulation quality. OBJECTIVE: Our study aimed to assess the TTR of VKAs in a hospitalized geriatric population and identify factors associated with low TTR. METHODS: This was a multicenter retrospective study of data from 1899 patients with a mean age of 87 years between 2013 and 2015 in the geriatric units of four French hospitals. The data collection consisted of 2450 VKA prescriptions. We excluded prescriptions with a duration of < 7 days, monitoring with fewer than two INR values and patients with prosthetic heart valves. TTR was assessed using the Rosendaal method. Factors associated with a low TTR (< 50%) were assessed using a non-parametric method. RESULTS: The mean TTR observed in this population was 42.6%. The TTR was < 50% for 62.5% of the patients included in this study. Significant associations were found between TTR < 50% and aspartate transaminase (AST), alkaline phosphatase (ALT), thyroid-stimulating hormone (TSH), prescription duration, fluconazole instauration, hemoglobin, and C-reactive protein (CRP). CONCLUSIONS: Both our results and those in the literature indicate that TTR in geriatric populations is lower than that in the general population. Most patients had an insufficient TTR, exposing them to an increased risk of thromboembolic and hemorrhagic events. These data provide a perspective on poor-quality anticoagulation and illustrates the difficulty of using VKAs in geriatric patients.

A new role for Holliday junction resolvase Yen1 in processing DNA replication intermediates exposes Dna2 as an accessory replicative helicase.

DNA replication is mediated by a multi-protein complex known as the replisome. With the hexameric MCM (minichromosome maintenance) replicative helicase at its core, the replisome splits the parental DNA strands, forming replication forks (RFs), where it catalyses coupled leading and lagging strand DNA synthesis. While replication is a highly effective process, intrinsic and oncogene-induced replication stress impedes the progression of replisomes along chromosomes. As a consequence, RFs stall, arrest, and collapse, jeopardizing genome stability. In these instances, accessory fork progression and repair factors, orchestrated by the replication checkpoint, promote RF recovery, ensuring the chromosomes are fully replicated and can be safely segregated at cell division. Homologous recombination (HR) proteins play key roles in negotiating replication stress, binding at stalled RFs and shielding them from inappropriate processing. In addition, HR-mediated strand exchange reactions restart stalled or collapsed RFs and mediate error-free post-replicative repair. DNA transactions at stalled RFs further involve various DNA editing factors, notably helicases and nucleases. A study by Ölmezer et al. (2016) has recently identified a role for the structure-specific nuclease Yen1 (GEN1 in human) in the resolution of dead-end DNA replication intermediates after RF arrest. This new function of Yen1 is distinct from its previously known role as a Holliday junction resolvase, mediating the removal of branched HR intermediates, and it becomes essential for viable chromosome segregation in cells with a defective Dna2 helicase. These findings have revealed greater complexity in the tasks mediated by Yen1 and expose a replicative role for the elusive helicase activity of the conserved Dna2 nuclease-helicase.

Replication intermediates that escape Dna2 activity are processed by Holliday junction resolvase Yen1.

Cells have evolved mechanisms to protect, restart and repair perturbed replication forks, allowing full genome duplication, even under replication stress. Interrogating the interplay between nuclease-helicase Dna2 and Holliday junction (HJ) resolvase Yen1, we find the Dna2 helicase activity acts parallel to homologous recombination (HR) in promoting DNA replication and chromosome detachment at mitosis after replication fork stalling. Yen1, but not the HJ resolvases Slx1-Slx4 and Mus81-Mms4, safeguards chromosome segregation by removing replication intermediates that escape Dna2. Post-replicative DNA damage checkpoint activation in Dna2 helicase-defective cells causes terminal G2/M arrest by precluding Yen1-dependent repair, whose activation requires progression into anaphase. These findings explain the exquisite replication stress sensitivity of Dna2 helicase-defective cells, and identify a non-canonical role for Yen1 in the processing of replication intermediates that is distinct from HJ resolution. The involvement of Dna2 helicase activity in completing replication may have implications for DNA2-associated pathologies, including cancer and Seckel syndrome.

Reversal of the DNA-binding-induced loop L1 conformational switch in an engineered human p53 protein.

The gene encoding the p53 tumor suppressor protein, a sequence-specific DNA binding transcription factor, is the most frequently mutated gene in human cancer. Crystal structures of homo-oligomerizing p53 polypeptides with specific DNA suggest that DNA binding is associated with a conformational switch. Specifically, in the absence of DNA, loop L1 of the p53 DNA binding domain adopts an extended conformation, whereas two p53 subunits switch to a recessed loop L1 conformation when bound to DNA as a tetramer. We previously designed a p53 protein, p53FG, with amino substitutions S121F and V122G targeting loop L1. These two substitutions enhanced the affinity of p53 for specific DNA yet, counterintuitively, decreased the residency time of p53 on DNA. Here, we confirmed these DNA binding properties of p53FG using a different method. We also determined by crystallography the structure of p53FG in its free state and bound to DNA as a tetramer. In the free state, loop L1 adopted a recessed conformation, whereas upon DNA binding, two subunits switched to the extended loop L1 conformation, resulting in a final structure that was very similar to that of wild-type p53 bound to DNA. Thus, altering the apo structure of p53 changed its DNA binding properties, even though the DNA-bound structure was not altered.