Comparison of the qCON and qNOX indices for the assessment of unconsciousness level and noxious stimulation response during surgery

The objective of this work is to compare the performances of two electroencephalogram based indices for detecting loss of consciousness and loss of response to nociceptive stimulation. Specifically, their behaviour after drug induction and during recovery of consciousness was pointed out. Data was recorded from 140 patients scheduled for general anaesthesia with a combination of propofol and remifentanil. The qCON 2000 monitor (Quantium Medical, Barcelona, Spain) was used to calculate the qCON and qNOX. Loss of response to verbal command and loss of eye-lash reflex were assessed during the transition from awake to anesthetized, defining the state of loss of consciousness. Movement as a response to laryngeal mask (LMA) insertion was interpreted as the response to the nociceptive stimuli. The patients were classified as movers or non-movers. The values of qCON and qNOX were statistically compared. Their fall times and rise times defined at the start and at the end of the surgery were calculated and compared. The results showed that the qCON was able to predict loss of consciousness such as loss of verbal command and eyelash reflex better than qNOX, while the qNOX has a better predictive value for response to noxious stimulation such as LMA insertion. From the analysis of the fall and rise times, it was found that the qNOX fall time (median: 217 s) was significantly longer (p value <0.05) than the qCON fall time (median: 150 s). At the end of the surgery, the qNOX started to increase in median at 45 s before the first annotation related to response to stimuli or recovery of consciousness, while the qCON at 88 s after the first annotation related to response to stimuli or recovery of consciousness (p value <0.05). The indices qCON and qNOX showed different performances in the detection of loss of consciousness and loss of response to stimuli during induction and recovery of consciousness. Furthermore, the qCON showed faster decrease during induction. This behaviour is associated with the hypothesis that the loss of response to stimuli (analgesic effect) might be reached after the loss of consciousness (hypnotic effect). On the contrary, the qNOX showed a faster increase at the end of the surgery, associated with the hypothesis that a higher probability of response to stimuli might be reached before the recovery of consciousness.Postprint (author's final draft

Refined multiscale entropy using fuzzy metrics: validation and application to nociception assessmentt

The refined multiscale entropy (RMSE) approach is commonly applied to assess complexity as a function of the time scale. RMSE is normally based on the computation of sample entropy (SampEn) estimating complexity as conditional entropy. However, SampEn is dependent on the length and standard deviation of the data. Recently, fuzzy entropy (FuzEn) has been proposed, including several refinements, as an alternative to counteract these limitations. In this work, FuzEn, translated FuzEn (TFuzEn), translated-reflected FuzEn (TRFuzEn), inherent FuzEn (IFuzEn), and inherent translated FuzEn (ITFuzEn) were exploited as entropy-based measures in the computation of RMSE and their performance was compared to that of SampEn. FuzEn metrics were applied to synthetic time series of different lengths to evaluate the consistency of the different approaches. In addition, electroencephalograms of patients under sedation-analgesia procedure were analyzed based on the patient’s response after the application of painful stimulation, such as nail bed compression or endoscopy tube insertion. Significant differences in FuzEn metrics were observed over simulations and real data as a function of the data length and the pain responses. Findings indicated that FuzEn, when exploited in RMSE applications, showed similar behavior to SampEn in long series, but its consistency was better than that of SampEn in short series both over simulations and real data. Conversely, its variants should be utilized with more caution, especially whether processes exhibit an important deterministic component and/or in nociception prediction at long scalesPeer ReviewedPostprint (published version

Poincaré plot analysis of cerebral blood flow signals : feature extraction and classification methods for apnea detection

Objective: Rheoencephalography is a simple and inexpensive technique for cerebral blood flow assessment, however, it is not used in clinical practice since its correlation to clinical conditions has not yet been extensively proved. The present study investigates the ability of Poincaré Plot descriptors from rheoencephalography signals to detect apneas in volunteers. Methods:A group of 16 subjects participated in the study. Rheoencephalography data from baseline and apnea periods were recorded and Poincaré Plot descriptors were extracted from the reconstructed attractors with different time lags (t). Among the set of extracted features, those presenting significant differences between baseline and apnea recordings were used as inputs to four different classifiers to optimize the apnea detection. Results:Three features showed significant differences between apnea and baseline signals: the Poincaré Plot ratio (SDratio), its correlation (R) and the Complex Correlation Measure (CCM). Those differences were optimized for time lags smaller than those recommended in previous works for other biomedical signals, all of them being lower than the threshold established by the position of the inflection point in the CCM curves. The classifier showing the best performance was the classification tree, with 81% accuracy and an area under the curve of the receiver operating characteristic of 0.927. This performance was obtained using a single input parameter, either SDratio or R. Conclusions Poincaré Plot features extracted from the attractors of rheoencephalographic signals were able to track cerebral blood flow changes provoked by breath holding. Even though further validation with independent datasets is needed, those results suggest that nonlinear analysis of rheoencephalography might be a useful approach to assess the correlation of cerebral impedance with clinical changesPeer ReviewedPostprint (published version

Drf1-dependent Kinase Interacts with Claspin through a Conserved Protein Motif

The Dbf4/Drf1-dependent kinase (DDK) is required for the initiation of DNA replication in eukaryotes. Another protein, Claspin, mediates the activation of a cellular checkpoint response to stalled replication forks and is also a regulator of replication. In this study, we found that DDK phosphorylates Claspin in vitro and forms a nuclear complex containing Cdc7, Drf1, and Claspin in Xenopus egg extracts. In addition, purified Claspin and DDK are capable of a direct in vitro interaction. We identified a conserved binding site on Claspin required for its interaction with DDK. This site corresponds to the first of two sequence repeats in the Chk1-binding domain of Claspin. Furthermore, we have established that two amino acids in this motif, Asp^(861) and Gln^(866), are essential for the interaction between Claspin and DDK. We found that mutant forms of Claspin incapable of interacting with DDK are still able to associate with and activate Chk1 in response to DNA replication blockages. However, Claspin-depleted egg extracts that have been reconstituted with these mutants of Claspin undergo DNA replication more slowly. These findings suggest that the interaction of DDK with Claspin mediates a checkpoint-independent function of Claspin related to DNA replication

The dynamics of replication licensing in live Caenorhabditis elegans embryos

Accurate DNA replication requires proper regulation of replication licensing, which entails loading MCM-2-7 onto replication origins. In this paper, we provide the first comprehensive view of replication licensing in vivo, using video microscopy of Caenorhabditis elegans embryos. As expected, MCM-2-7 loading in late M phase depended on the prereplicative complex (pre-RC) proteins: origin recognition complex (ORC), CDC-6, and CDT-1. However, many features we observed have not been described before: GFP-ORC-1 bound chromatin independently of ORC-2-5, and CDC-6 bound chromatin independently of ORC, whereas CDT-1 and MCM-2-7 DNA binding was interdependent. MCM-3 chromatin loading was irreversible, but CDC-6 and ORC turned over rapidly, consistent with ORC/CDC-6 loading multiple MCM-2-7 complexes. MCM-2-7 chromatin loading further reduced ORC and CDC-6 DNA binding. This dynamic behavior creates a feedback loop allowing ORC/CDC-6 to repeatedly load MCM-2-7 and distribute licensed origins along chromosomal DNA. During S phase, ORC and CDC-6 were excluded from nuclei, and DNA was overreplicated in export-defective cells. Thus, nucleocytoplasmic compartmentalization of licensing factors ensures that DNA replication occurs only once

The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation.

The replicative mini-chromosome-maintenance 2-7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the 'initial' complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 and is salt-sensitive, classifying the arrested complex as a helicase-loading intermediate. Accordingly, we found that overexpression of the mutants cause cell-cycle arrest and dominant lethality. Our work identifies the OCM complex as competent for MCM2-7 dimerization, reveals MCM2-7 dimerization as a limiting step during pre-RC formation and defines critical mechanisms that explain how origins are licensed

Xenopus Mcm10 is a CDK-substrate required for replication fork stability

<p>During S phase, following activation of the S phase CDKs and the DBF4-dependent kinases (DDK), double hexamers of Mcm2-7 at licensed replication origins are activated to form the core replicative helicase. Mcm10 is one of several proteins that have been implicated from work in yeasts to play a role in forming a mature replisome during the initiation process. Mcm10 has also been proposed to play a role in promoting replisome stability after initiation has taken place. The role of Mcm10 is particularly unclear in metazoans, where conflicting data has been presented. Here, we investigate the role and regulation of Mcm10 in <i>Xenopus</i> egg extracts. We show that <i>Xenopus</i> Mcm10 is recruited to chromatin late in the process of replication initiation and this requires prior action of DDKs and CDKs. We also provide evidence that Mcm10 is a CDK substrate but does not need to be phosphorylated in order to associate with chromatin. We show that in extracts depleted of more than 99% of Mcm10, the bulk of DNA replication still occurs, suggesting that Mcm10 is not required for the process of replication initiation. However, in extracts depleted of Mcm10, the replication fork elongation rate is reduced. Furthermore, the absence of Mcm10 or its phosphorylation by CDK results in instability of replisome proteins on DNA, which is particularly important under conditions of replication stress.</p

Ufd1-Npl4 recruit Cdc48 for disassembly of ubiquitylated CMG helicase at the end of chromosome replication

Disassembly of the Cdc45-MCM-GINS (CMG) DNA helicase is the key regulated step during DNA replication termination in eukaryotes, involving ubiquitylation of the Mcm7 helicase subunit, leading to a disassembly process that requires the Cdc48 “segregase”. Here, we employ a screen to identify partners of budding yeast Cdc48 that are important for disassembly of ubiquitylated CMG helicase at the end of chromosome replication. We demonstrate that the ubiquitin-binding Ufd1-Npl4 complex recruits Cdc48 to ubiquitylated CMG. Ubiquitylation of CMG in yeast cell extracts is dependent upon lysine 29 of Mcm7, which is the only detectable site of ubiquitylation both in vitro and in vivo (though in vivo other sites can be modified when K29 is mutated). Mutation of K29 abrogates in vitro recruitment of Ufd1-Npl4-Cdc48 to the CMG helicase, supporting a model whereby Ufd1-Npl4 recruits Cdc48 to ubiquitylated CMG at the end of chromosome replication, thereby driving the disassembly reaction

Regulation of Unperturbed DNA Replication by Ubiquitylation

Posttranslational modification of proteins by means of attachment of a small globular protein ubiquitin (i.e., ubiquitylation) represents one of the most abundant and versatile mechanisms of protein regulation employed by eukaryotic cells. Ubiquitylation influences almost every cellular process and its key role in coordination of the DNA damage response is well established. In this review we focus, however, on the ways ubiquitylation controls the process of unperturbed DNA replication. We summarise the accumulated knowledge showing the leading role of ubiquitin driven protein degradation in setting up conditions favourable for replication origin licensing and S-phase entry. Importantly, we also present the emerging major role of ubiquitylation in coordination of the active DNA replication process: preventing re-replication, regulating the progression of DNA replication forks, chromatin re-establishment and disassembly of the replisome at the termination of replication forks