Analysis by confocal microscopy of the behavior of heat shock protein 70 within the nucleus and of a nuclear matrix polypeptide during prolonged heat shock response in HeLa cells.

By means of confocal laser scanning microscopy and indirect fluorescence experiments we have examined the behavior of heat-shock protein 70 (HSP70) within the nucleus as well as of a nuclear matrix protein (M(r) = 125 kDa) during a prolonged heat-shock response (up to 24 h at 42 degrees C) in HeLa cells. In control cells HSP70 was mainly located in the cytoplasm. The protein translocated within the nucleus upon cell exposure to hyperthermia. The fluorescent pattern revealed by monoclonal antibody to HSP70 exhibited several changes during the 24-h-long incubation. The nuclear matrix protein showed changes in its location that were evident as early as 1 h after initiation of heat shock. After 7 h of treatment, the protein regained its original distribution. However, in the late stages of the hyperthermic treatment (17-24 h) the fluorescent pattern due to 125-kDa protein changed again and its original distribution was never observed again. These results show that HSP70 changes its localization within the nucleus conceivably because it is involved in solubilizing aggregated polypeptides present in different nuclear regions. Our data also strengthen the contention that proteins of the insoluble nucleoskeleton are involved in nuclear structure changes that occur during heat-shock response.

A comparison of methods for differential expression analysis of RNA-seq data.

BACKGROUND: Finding genes that are differentially expressed between conditions is an integral part of understanding the molecular basis of phenotypic variation. In the past decades, DNA microarrays have been used extensively to quantify the abundance of mRNA corresponding to different genes, and more recently high-throughput sequencing of cDNA (RNA-seq) has emerged as a powerful competitor. As the cost of sequencing decreases, it is conceivable that the use of RNA-seq for differential expression analysis will increase rapidly. To exploit the possibilities and address the challenges posed by this relatively new type of data, a number of software packages have been developed especially for differential expression analysis of RNA-seq data. RESULTS: We conducted an extensive comparison of eleven methods for differential expression analysis of RNA-seq data. All methods are freely available within the R framework and take as input a matrix of counts, i.e. the number of reads mapping to each genomic feature of interest in each of a number of samples. We evaluate the methods based on both simulated data and real RNA-seq data. CONCLUSIONS: Very small sample sizes, which are still common in RNA-seq experiments, impose problems for all evaluated methods and any results obtained under such conditions should be interpreted with caution. For larger sample sizes, the methods combining a variance-stabilizing transformation with the 'limma' method for differential expression analysis perform well under many different conditions, as does the nonparametric SAMseq method.

Impact of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry on the Clinical Management of Patients With Gram-negative Bacteremia: A Prospective Observational Study.

Background. Early identification of pathogens from blood cultures using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry may optimize the choice of empirical antibiotic therapy in the setting of bloodstream infections. We aimed to assess the impact of this new technology on the use of antibiotic treatment in patients with gram-negative bacteremia. Methods. We conducted a prospective observational study from January to December 2010 to evaluate the sequential and separate impacts of Gram stain reporting and MALDI-TOF bacterial identification performed on blood culture pellets in patients with gram-negative bacteremia. The primary outcome was the impact of MALDI-TOF on empirical antibiotic choice. Results. Among 202 episodes of gram-negative bacteremia, Gram stain reporting had an impact in 42 cases (20.8%). MALDI-TOF identification led to a modification of empirical therapy in 71 of all 202 cases (35.1%), and in 16 of 27 cases (59.3%) of monomicrobial bacteremia caused by AmpC-producing Enterobacteriaceae. The most frequently observed impact was an early appropriate broadening of the antibiotic spectrum in 31 of 71 cases (43.7%). In total, 143 of 165 episodes (86.7%) of monomicrobial bacteremia were correctly identified at genus level by MALDI-TOF. Conclusions. In a low prevalence area for extended spectrum betalactamases (ESBL) and multiresistant gram-negative bacteria, MALDI-TOF performed on blood culture pellets had an impact on the clinical management of 35.1% of all gram-negative bacteremia cases, demonstrating a greater impact than Gram stain reporting. Thus, MALDI-TOF could become a vital second step beside Gram stain in guiding the empirical treatment of patients with bloodstream infection.

Molecular imaging of matrix metalloproteinases in atherosclerotic plaques.

Ischaemic stroke and myocardial infarction often result from the sudden rupture of an atherosclerotic plaque. The subsequent arterial thrombosis occluding the vessel lumen has been widely indicated as the crucial acute event causing peripheral tissue ischaemia. A complex cross-talk between systemic and intraplaque inflammatory mediators has been shown to regulate maturation, remodeling and final rupture of an atherosclerotic plaque. Matrix metalloproteinases (MMPs) are proteolytic enzymes (released by several cell subsets within atherosclerotic plaques), which favour atherogenesis and increase plaque vulnerability. Thus, the assessment of intraplaque levels and activity of MMP might be of pivotal relevance in the evaluation of the risk of rupture. New imaging approaches, focused on the visualisation of inflammation in the vessel wall and plaque, may emerge as tools for individualised risk assessment and prevention of events. In this review, we summarize experimental findings of the currently available invasive and noninvasive imaging techniques, used to detect the presence and activity of MMPs in atherosclerotic plaques.

A program for the computation of power and determination of sample size in hierarchical experimental designs.

We have devised a program that allows computation of the power of F-test, and hence determination of appropriate sample and subsample sizes, in the context of the one-way hierarchical analysis of variance with fixed effects. The power at a fixed alternative is an increasing function of the sample size and of the subsample size. The program makes it easy to obtain the power of F-test for a range of values of sample and subsample sizes, and therefore the appropriate sizes based on a desired power. The program can be used for the 'ordinary' case of the one-way analysis of variance, as well as for hierarchical analysis of variance with two stages of sampling. Examples are given of the practical use of the program.

Using matrix attachment regions to improve recombinant protein production.

Chinese hamster ovary (CHO) cells are the system of choice for the production of complex molecules, such as monoclonal antibodies. Despite significant progress in improving the yield from these cells, the process to the selection, identification, and maintenance of high-producing cell lines remains cumbersome, time consuming, and often of uncertain outcome. Matrix attachment regions (MARs) are DNA sequences that help generate and maintain an open chromatin domain that is favourable to transcription and may also facilitate the integration of several copies of the transgene. By incorporating MARs into expression vectors, an increase in the proportion of high-producer cells as well as an increase in protein production are seen, thereby reducing the number of clones to be screened and time to production by as much as 9 months. In this chapter, we describe how MARs can be used to increase transgene expression and provide protocols for the transfection of CHO cells in suspension and detection of high-producing antibody cell clones.

Psychometric properties of the french translation of the constructive thinking inventory in a sample of adolescents and young adults

The Constructive Thinking Inventory (CTI) measures cognitive coping strategies used in everyday problem solving. The main objective of this study was to assess the factorial structure, the internal consistency, the correspondence with the American normative values, and the discriminant validity of the French translation. A community sample of 777 students aged 12 to 26 years, recruited from schools, colleges and universities, answered the 108item selfreport CTI questionnaire during a class period. A sample of 60 male adolescent offenders aged 13 to 18 years, recruited from two institutions for juvenile offenders, answered the CTI during an individual interview. Results show that the French translation of the CTI follows an identical factorial structure as the Epstein's American version in both adolescents and young adults, and that its internal consistency is satisfactory. Differences in Constructive Thinking profiles according to gender and age and between Swiss and American samples, are discussed. Juvenile offenders differed from community youths on most of the scales, speaking for a good discriminant validity of the CTI. In conclusion, the French translation of the CTI appears to preserve the original version's psychometric properties. The present study provides normative values from a community sample of Swiss adolescents and young adults.

Matrix metalloproteinase 9 (MMP-9/gelatinase B) proteolytically cleaves ICAM-1 and participates in tumor cell resistance to natural killer cell-mediated cytotoxicity.

Shedding of intercellular adhesion molecule 1 (ICAM-1) is believed to play a role in tumor cell resistance to cell-mediated cytotoxicity. However, the mechanism whereby ICAM-1 is shed from the surface of tumor cells remains unclear. In this study, we have addressed the possibility that matrix metalloproteinases are implicated in ICAM-1 shedding. Our observations suggest a functional relationship between ICAM-1 and matrix metalloproteinase 9 (MMP-9) whereby ICAM-1 provides a cell surface docking mechanism for proMMP-9, which, upon activation, proteolytically cleaves the extracellular domain of ICAM-1 leading to its release from the cell surface. MMP-9-dependent shedding of ICAM-1 is found to augment tumor cell resistance to natural killer (NK) cell-mediated cytotoxicity. Taken together, our observations propose a mechanism for ICAM-1 shedding from the cell surface and provide support for MMP involvement in tumor cell evasion of immune surveillance.

Circulating matrix γ-carboxyglutamate protein (MGP) species are refractory to vitamin K treatment in a new case of Keutel syndrome.

Summary.  Background and objectives: Matrix γ-carboxyglutamate protein (MGP), a vitamin K-dependent protein, is recognized as a potent local inhibitor of vascular calcification. Studying patients with Keutel syndrome (KS), a rare autosomal recessive disorder resulting from MGP mutations, provides an opportunity to investigate the functions of MGP. The purpose of this study was (i) to investigate the phenotype and the underlying MGP mutation of a newly identified KS patient, and (ii) to investigate MGP species and the effect of vitamin K supplements in KS patients. Methods: The phenotype of a newly identified KS patient was characterized with specific attention to signs of vascular calcification. Genetic analysis of the MGP gene was performed. Circulating MGP species were quantified and the effect of vitamin K supplements on MGP carboxylation was studied. Finally, we performed immunohistochemical staining of tissues of the first KS patient originally described focusing on MGP species. Results: We describe a novel homozygous MGP mutation (c.61+1G>A) in a newly identified KS patient. No signs of arterial calcification were found, in contrast to findings in MGP knockout mice. This patient is the first in whom circulating MGP species have been characterized, showing a high level of phosphorylated MGP and a low level of carboxylated MGP. Contrary to expectations, vitamin K supplements did not improve the circulating carboxylated MGP levels. Phosphorylated MGP was also found to be present in the first KS patient originally described. Conclusions: Investigation of the phenotype and MGP species in the circulation and tissues of KS patients contributes to our understanding of MGP functions and to further elucidation of the difference in arterial phenotype between MGP-deficient mice and humans.

Measurements of seismic attenuation and transient fluid pressure in partially saturated Berea sandstone: Evidence of fluid flow on the mesoscopic scale

A novel laboratory technique is proposed to investigate wave-induced fluid flow on the mesoscopic scale as a mechanism for seismic attenuation in partially saturated rocks. This technique combines measurements of seismic attenuation in the frequency range from 1 to 100?Hz with measurements of transient fluid pressure as a response of a step stress applied on top of the sample. We used a Berea sandstone sample partially saturated with water. The laboratory results suggest that wave-induced fluid flow on the mesoscopic scale is dominant in partially saturated samples. A 3-D numerical model representing the sample was used to verify the experimental results. Biot's equations of consolidation were solved with the finite-element method. Wave-induced fluid flow on the mesoscopic scale was the only attenuation mechanism accounted for in the numerical solution. The numerically calculated transient fluid pressure reproduced the laboratory data. Moreover, the numerically calculated attenuation, superposed to the frequency-independent matrix anelasticity, reproduced the attenuation measured in the laboratory in the partially saturated sample. This experimental?numerical fit demonstrates that wave-induced fluid flow on the mesoscopic scale and matrix anelasticity are the dominant mechanisms for seismic attenuation in partially saturated Berea sandstone.

Multi-level modeling of aspects associated with poor mental health in a sample of prehospital emergency professionals.

BACKGROUND: The goal of this paper is to investigate the respective influence of work characteristics, the effort-reward ratio, and overcommitment on the poor mental health of out-of-hospital care providers. METHODS: 333 out-of-hospital care providers answered a questionnaire that included queries on mental health (GHQ-12), demographics, health-related information and work characteristics, questions from the Effort-Reward Imbalance Questionnaire, and items about overcommitment. A two-level multiple regression was performed between mental health (the dependent variable) and the effort-reward ratio, the overcommitment score, weekly number of interventions, percentage of non-prehospital transport of patients out of total missions, gender, and age. Participants were first-level units, and ambulance services were second-level units. We also shadowed ambulance personnel for a total of 416 hr. RESULTS: With cutoff points of 2/3 and 3/4 positive answers on the GHQ-12, the percentages of potential cases with poor mental health were 20% and 15%, respectively. The effort-reward ratio was associated with poor mental health (P < 0.001), irrespective of age or gender. Overcommitment was associated with poor mental health; this association was stronger in women (β = 0.054) than in men (β = 0.020). The percentage of prehospital missions out of total missions was only associated with poor mental health at the individual level. CONCLUSIONS: Emergency medical services should pay attention to the way employees perceive their efforts and the rewarding aspects of their work: an imbalance of those aspects is associated with poor mental health. Low perceived esteem appeared particularly associated with poor mental health. This suggests that supervisors of emergency medical services should enhance the value of their employees' work. Employees with overcommitment should also receive appropriate consideration. Preventive measures should target individual perceptions of effort and reward in order to improve mental health in prehospital care providers.

Socioeconomics status and quality of care in a population-based sample of swiss diabetic patients

Introduction: Low socioeconomic status (SES) is associated with higher prevalence of diabetes and worse outcomes; it has also been shown to be associated with worse quality of care. We aimed to explore the relationship between SES and quality of care in the Swiss context. Methods: We used data from a population-based survey including 519 adult diabetic patients living in the canton of Vaud. Self-reported data on patients' and diabetes characteristics, indicators of process and outcomes of care and quality of life were collected. Dependent variables included 6 processes of care (PoC) received during the last 12 months (HbA1C, lipid, microalbuminuria, fundoscopy, feet examination and influenza vaccination) and selected clinical outcomes (blood pressure, LDL, HbA1C, diabetes-specific (ADDQoL) and generic quality of life (SF-12)). Regression analyses were performed to assess the relationship between education and income, respectively, and quality of care as measured by PoC and clinical outcomes. Adjustment was made for age, gender and comorbidities. Results: Mean age was 64.5 years, 40% were women; 19%, 56% and 25% of the patients reported primary (I), secondary (II) and tertiary (III) education. Fundoscopy was the only PoC significantly associated with education, with III education patients more likely to get the exam than those with primary education (adjOR 1.8, 95% CI 1.0-3.3). Use of composite indicators of PoC showed that compared to patients with primary education, patients with III education were more likely to receive ≥5/6 PoC (adjOR 1.9, 95% CI 1.1-3.4), and that those with II or III education were more likely to receive 4/4 PoC (adjOR 1.9, 95% CI 1.0-3.3; adjOR 2.1, 95% CI 1.1-4.1, respectively). Quality of life was the only clinical outcome significantly associated with education, with II and III education patients reporting better quality of life compared to primary education patients, as measured by the ADDQoL (β 0.6, 95% CI 0.3-1.0, β 0.6, 95% CI 0.2-1.0, respectively) and the physical component score of the SF-12 (β 2.5, 95% CI 0.2-4.8, β 3.6, 95% CI 0.9-6.4, respectively). No associations were found between income and quality of care. Conclusion: Social inequalities have been demonstrated in Switzerland for global health indicators. Our results suggest that similar associations are found when considering quality of care measures in individuals with diabetes, but only for a few indicators.

Simultaneous quantification of selective serotonin reuptake inhibitors and metabolites in human plasma by liquid chromatography-electrospray mass spectrometry for therapeutic drug monitoring.

A simple and sensitive liquid chromatography-electrospray ionization mass spectrometry method was developed for the simultaneous quantification in human plasma of all selective serotonin reuptake inhibitors (citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline) and their main active metabolites (desmethyl-citalopram and norfluoxetine). A stable isotope-labeled internal standard was used for each analyte to compensate for the global method variability, including extraction and ionization variations. After sample (250μl) pre-treatment with acetonitrile (500μl) to precipitate proteins, a fast solid-phase extraction procedure was performed using mixed mode Oasis MCX 96-well plate. Chromatographic separation was achieved in less than 9.0min on a XBridge C18 column (2.1×100mm; 3.5μm) using a gradient of ammonium acetate (pH 8.1; 50mM) and acetonitrile as mobile phase at a flow rate of 0.3ml/min. The method was fully validated according to Société Française des Sciences et Techniques Pharmaceutiques protocols and the latest Food and Drug Administration guidelines. Six point calibration curves were used to cover a large concentration range of 1-500ng/ml for citalopram, desmethyl-citalopram, paroxetine and sertraline, 1-1000ng/ml for fluoxetine and fluvoxamine, and 2-1000ng/ml for norfluoxetine. Good quantitative performances were achieved in terms of trueness (84.2-109.6%), repeatability (0.9-14.6%) and intermediate precision (1.8-18.0%) in the entire assay range including the lower limit of quantification. Internal standard-normalized matrix effects were lower than 13%. The accuracy profiles (total error) were mainly included in the acceptance limits of ±30% for biological samples. The method was successfully applied for routine therapeutic drug monitoring of more than 1600 patient plasma samples over 9 months. The β-expectation tolerance intervals determined during the validation phase were coherent with the results of quality control samples analyzed during routine use. This method is therefore precise and suitable both for therapeutic drug monitoring and pharmacokinetic studies in most clinical laboratories.