Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.

BACKGROUND: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades. RESULTS: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage. CONCLUSIONS: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements.

Population Variation and Genetic Control of Modular Chromatin Architecture in Humans.

Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain.

Uncovering Cryptic Parasitoid Diversity in Horismenus (Chalcidoidea, Eulophidae).

Horismenus parasitoids are an abundant and understudied group of eulophid wasps found mainly in the New World. Recent surveys based on morphological analyses in Costa Rica have quadrupled the number of named taxa, with more than 400 species described so far. This recent revision suggests that there is still a vast number of unknown species to be identified. As Horismenus wasps have been widely described as parasitoids of insect pests associated with crop plants, it is of high importance to properly establish the extant diversity of the genus, in order to provide biological control practitioners with an exhaustive catalog of putative control agents. In this study, we first collected Horismenus wasps from wild Phaseolus bean seeds in Central Mexico and Arizona to assess the genetic relatedness of three morphologically distinct species with overlapping host and geographical ranges. Sequence data from two nuclear and two mitochondrial gene regions uncovered three cryptic species within each of the three focal species (i.e., H. missouriensis, H. depressus and H. butcheri). The monophyly of each cryptic group is statistically supported (except in two of them represented by one single tip in which monophyly cannot be tested). The phylogenetic reconstruction is discussed with respect to differences between gene regions as well as likely reasons for the differences in variability between species.

Practice variability and efficacy of clonazepam, lorazepam, and midazolam in status epilepticus: A multicenter comparison.

OBJECTIVE: Benzodiazepines (BZD) are recommended as first-line treatment for status epilepticus (SE), with lorazepam (LZP) and midazolam (MDZ) being the most widely used drugs and part of current treatment guidelines. Clonazepam (CLZ) is also utilized in many countries; however, there is no systematic comparison of these agents for treatment of SE to date. METHODS: We identified all patients treated with CLZ, LZP, or MDZ as a first-line agent from a prospectively collected observational cohort of adult patients treated for SE in four tertiary care centers. Relative efficacies of CLZ, LZP, and MDZ were compared by assessing the risk of developing refractory SE and the number of antiseizure drugs (ASDs) required to control SE. RESULTS: Among 177 patients, 72 patients (40.62%) received CLZ, 82 patients (46.33%) LZP, and 23 (12.99%) MDZ; groups were similar in demographics and SE characteristics. Loading dose was considered insufficient in the majority of cases for LZP, with a similar rate (84%, 95%, and 87.5%) in the centers involved, and CLZ was used as recommended in 52% of patients. After adjustment for relevant variables, LZP was associated with an increased risk of refractoriness as compared to CLZ (odds ratio [OR] 6.4, 95% confidence interval [CI] 2.66-15.5) and with an increased number of ASDs needed for SE control (OR 4.35, 95% CI 1.8-10.49). SIGNIFICANCE: CLZ seems to be an effective alternative to LZP and MDZ. LZP is frequently underdosed in this setting. These findings are highly relevant, since they may impact daily practice.

Sources of variability of speech, spatial, and qualities of hearing scale (SSQ) scores in normal-hearing and hearing-impaired populations.

OBJECTIVE: To identify and quantify sources of variability in scores on the speech, spatial, and qualities of hearing scale (SSQ) and its short forms among normal-hearing and hearing-impaired subjects using a French-language version of the SSQ. DESIGN: Multi-regression analyses of SSQ scores were performed using age, gender, years of education, hearing loss, and hearing-loss asymmetry as predictors. Similar analyses were performed for each subscale (Speech, Spatial, and Qualities), for several SSQ short forms, and for differences in subscale scores. STUDY SAMPLE: One hundred normal-hearing subjects (NHS) and 230 hearing-impaired subjects (HIS). RESULTS: Hearing loss in the better ear and hearing-loss asymmetry were the two main predictors of scores on the overall SSQ, the three main subscales, and the SSQ short forms. The greatest difference between the NHS and HIS was observed for the Speech subscale, and the NHS showed scores well below the maximum of 10. An age effect was observed mostly on the Speech subscale items, and the number of years of education had a significant influence on several Spatial and Qualities subscale items. CONCLUSION: Strong similarities between SSQ scores obtained across different populations and languages, and between SSQ and short forms, underline their potential international use.

Typology of "Fatigue" by Heart Rate Variability Analysis in Elite Nordic-skiers.

This study investigated changes in heart rate variability (HRV) in elite Nordic-skiers to characterize different types of "fatigue" in 27 men and 30 women surveyed from 2004 to 2008. R-R intervals were recorded at rest during 8 min supine (SU) followed by 7 min standing (ST). HRV parameters analysed were powers of low (LF), high (HF) frequencies, (LF+HF) (ms(2)) and heart rate (HR, bpm). In the 1 063 HRV tests performed, 172 corresponded to a "fatigue" state and the first were considered for analysis. 4 types of "fatigue" (F) were identified: 1. F(HF(-)LF(-))SU_ST for 42 tests: decrease in LFSU (- 46%), HFSU (- 70%), LFST (- 43%), HFST (- 53%) and increase in HRSU (+ 15%), HRST (+ 14%). 2. F(LF(+) SULF(-) ST) for 8 tests: increase in LFSU (+ 190%) decrease in LFST (- 84%) and increase in HRST (+ 21%). 3. F(HF(-) SUHF(+) ST) for 6 tests: decrease in HFSU (- 72%) and increase in HFST (+ 501%). 4. F(HF(+) SU) for only 1 test with an increase in HFSU (+ 2161%) and decrease in HRSU (- 15%). Supine and standing HRV patterns were independently modified by "fatigue". 4 "fatigue"-shifted HRV patterns were statistically sorted according to differently paired changes in the 2 postures. This characterization might be useful for further understanding autonomic rearrangements in different "fatigue" conditions.

Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile.

Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.

Interrater variability of EEG interpretation in comatose cardiac arrest patients.

OBJECTIVE: EEG is widely used to predict outcome in comatose cardiac arrest patients, but its value has been limited by lack of a uniform classification. We used the EEG terminology proposed by the American Clinical Neurophysiology Society (ACNS) to assess interrater variability in a cohort of cardiac arrest patients included in the Target Temperature Management trial. The main objective was to evaluate if malignant EEG-patterns could reliably be identified. METHODS: Full-length EEGs from 103 comatose cardiac arrest patients were interpreted by four EEG-specialists with different nationalities who were blinded for patient outcome. Percent agreement and kappa (κ) for the categories in the ACNS EEG terminology and for prespecified malignant EEG-patterns were calculated. RESULTS: There was substantial interrater agreement (κ 0.71) for highly malignant patterns and moderate agreement (κ 0.42) for malignant patterns. Substantial agreement was found for malignant periodic or rhythmic patterns (κ 0.72) while agreement for identifying an unreactive EEG was fair (κ 0.26). CONCLUSIONS: The ACNS EEG terminology can be used to identify highly malignant EEG-patterns in post cardiac arrest patients in an international context with high reliability. SIGNIFICANCE: The establishment of strict criteria with high transferability between interpreters will increase the usefulness of routine EEG to assess neurological prognosis after cardiac arrest.

Heart Rate Variability (HRV) modifications in adult hemiplegic patients after botulinum toxin type A (nt-201) injection.

BACKGROUND: The most important adverse effect of BoNT-A is the systemic diffusion of the toxin. There is some evidence that the administration of high doses can increase the risk of systemic diffusion and the development of clinically evident adverse effects, however an international consensus does not exist about its maximum dose. AIM: The aim of this study was to evaluate changes in autonomic heart drive induced by high doses (higher than 600 units) of incobotulinumtoxinA injection in spastic stroke patients. Moreover, the treatment safety by monitoring adverse events occurrence was assessed. DESIGN: Case control study. POPULATION: Eleven stroke survivors with spastic hemiplegia. METHODS: Patients were treated with intramuscular focal injections of IncobotulinumtoxinA (NT 201; Xeomin®, Merz Pharmaceuticals GmbH, Frankfurt, Germany). Doses were below 12 units/Kg. Each patient underwent an ECG recording before injection and 10 days after treatment. Linear and non-linear Heart Rate variability (HRV) measures were derived from ECGs with a dedicated software. RESULTS: None of the variable considered showed statistically significant changes after BoNT-A injection. CONCLUSION: The use of incobotulinumtoxinA in adult patients at doses up to 12 units/kg seems to be safe regarding autonomic heart drive. CLINICAL REHABILITATION IMPACT: The use of IncobotulinumtoxinA up to 600 units could be a safe therapeutic option in spastic hemiplegic stroke survivors.