Serial protein labeling with infrared maleimide dyes to identify cysteine modifications

Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification and quantification of growing importance. However, saturation labeling of thiols with fluorescent dyes results in poor protein recuperation and therefore requires the use of large quantities of starting material. This is especially important in sequential dye-labeling steps when applied for an identification of cysteine modifications. First, we studied the effects of different detergents during labeling procedure, i.e. Tween 20, Triton X-100 and CHAPS, on protein yield and composition. Tween 20 and Triton X-100 resulted in yields of around 50% labeled proteins compared to only 10% with PBS alone and a most diversified 2-DE protein pattern. Secondly, Tween 20 was used for serial protein labeling with maleimid fluorophores, first to conjugate to accessible thiols and after a reduction to label with another fluorophore previously masked di-sulphide and/or oxidized proteins in frontal cortex autopsy tissue of a subject with mild Alzheimer's disease. Two-DE DIGE revealed a complex protein pattern of readily labeled thiols and di-sulphide and/or oxidized proteins. Seventeen proteins were identified by MALDI-TOF and by peptide fingerprints. Several proteins were oxidized and involved in Alzheimer's disease. However methionine oxidation was prevalent. Infrared DIGE may provide an additional tool for an identification of oxidation susceptible proteins.

Animal Tissue Response To Mid-Term Electronic Modular Artificial Sphincter Implantation

Introduction and objectives: The AMS 800TM is considered the gold standard for sphincter replacement. However, the one-ring design can erode the urethra and lead to severe complications. A mechanism that could alternatively compress successive segments of the urethra would limit such deleterious outcome. We report 12 weeks animal urethral tissue analysis following implantation of a new modular artificial sphincter. METHODS: The device is composed by three parts: the contractile unit, two rings and an integrated microprocessor. The contractile unit is made of Nitinol fibers. The rings are placed around the urethra to control the flow of urine by squeezing the urethra. They work in a sequential alternative mode and are controlled by a microprocessor connected to an external computer. The computer can reveal specific failure of device components. The device was impkanted in eight male sheep. The rings were positioned around the urethra and the control unit was placed 5cm away. The device was working twenty hours per day; it was open 10min. per hour to allow urination. The animals were sacrificed after 12 weeks. The urethra and the tissues surrounding the control unit were macroscopically and microscopically examined. Two transversal sections crossing the sphincter and two transversal sections crossing the urethra alone were obtained and stained with modified Paragon after resin embedding. Urethra was also embedded in paraffin. The first section was stained with safranin-hematoxylin-eosin, the second section was stained with Masson's Trichrome and the remaining eight sections were available for immunolabelling of the macrophages.Results: The chronic study went uneventful. No clinical infection or pain was observed. The computer registered no specific failure in ring function, Nitinol wires and tube connectors. At explantation, except for a slight grade of lymphocytes in two out of eight specimens, no urethral stricture or atrophy could be observed. Immunohistochemistry confirmed the absence of macrophages. Tissue structure and organization of the urethra with and without artificial sphincter were similar. No migration of the device was observed.Conclusions: The study clearly showed no tissue damage or inflammation of the urethra. Electronic design, preservation of urethral vascularisation and adjustability after implantation are the key ideas to improve the actual AUS. Further studies will be carried out to evaluate this potential.

Le traitement de l'hypertension artérielle: quel médicament pour quelle malade [The treatment of arterial hypertension: which drug for which patient?]

Hypertension is a multifactorial disease. Various antihypertensive drugs can lower arterial pressure in a given patient in a more or less efficient way. The sequential testing of several drugs is most promising for lowering blood pressure by monotherapy. If necessary a drug combination is preferable to dose adjustments of a single substance because of the risk for side effects growing with the dose.

Typing Candida albicans oral isolates from human immunodeficiency virus-infected patients by multilocus enzyme electrophoresis and DNA fingerprinting.

A total of 189 Candida albicans isolates have been typed by multilocus enzyme electrophoresis. The results obtained confirm the clonal mode of reproduction of C. albicans. The C. albicans populations found in the oropharynx of human immunodeficiency virus (HIV)-infected patients, in the oropharynx of healthy carriers, or in association with invasive candidiasis could not be distinguished. No clone or group of clones could be associated with the appearance of clinical disorders or with a reduced in vitro susceptibility to the antifungal agent fluconazole. Multiple and sequential oral isolates from 24 HIV-infected patients were also typed by restriction enzyme analysis with the enzymes EcoRI and HinfI and by use of the Ca3 repetitive probe. The results obtained by the combination of all three typing methods show that all but one patient each carried a unique major C. albicans clone in their oropharynx. The 21 patients with sequential isolates had the same C. albicans clones in their throats during recurrent oropharyngeal candidiasis episodes, independently of clinical status or of changes of in vitro susceptibility to fluconazole. Finally, several isolates of the same C. albicans clone found simultaneously in the oropharynx of a patient may present different levels of susceptibility to fluconazole.

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.

Cancers du sein : comment associer l'hormonothérapie et la radiothérapie en situation adjuvante ? [How to combine hormonotherapy and radiation treatment in adjuvant breast cancer ?]

L'hormonoradiothérapie concomitante est utilisée depuis plusieurs années en pratique clinique quotidienne dans les cancers localement évolués de la prostate. Le transfert de ce concept en pathologie mammaire a été très peu rapporté dans la littérature, mais semble pourtant licite devant l'hormonodépendance fréquente des cancers du sein et la synergie potentielle de ces deux armes thérapeutiques. En situation adjuvante, deux stratégies sont actuellement utilisées : la prescription d'un inhibiteur de l'aromatase d'emblée ou après un délai plus ou moins long de tamoxifène. En pratique, ces molécules peuvent donc interagir avec la radiothérapie adjuvante. Les études rétrospectives récemment publiées n'ont pas mis en évidence de différence significative sur l'incidence des évènements, notamment locorégionaux, de l'association concomitante ou séquentielle du tamoxifène à la radiothérapie. La toxicité de l'association reste discutable en termes de fibroses sous-cutanée et pulmonaire. Il semble que le tamoxifène aggraverait les séquelles postradiques uniquement chez les patientes prédisposées à souffrir d'effets tardifs de la radiothérapie et identifiées par un test prédictif biologique. La prudence reste donc encore de mise du moins pour ces patientes. Cet article détaille les avantages et les risques de l'utilisation concomitante de la radiothérapie et de l'hormonothérapie adjuvantes des cancers localisés du sein. Combined radiation and hormone therapies have become common clinical practice in recent years for locally-advanced prostate cancers. The use of such concomitant therapy in the treatment of breast disease has been infrequently reported in the literature, but seems justified given the common hormonal dependence of breast cancer and the potential synergistic effect of these two treatment modalities. As adjuvant therapy, two strategies are used in daily clinical practice: upfront aromatase inhibitors or sequentially after a variable delay of tamoxifen. These molecules may, thus, interact with radiotherapy. Retrospectives studies recently published did not show any differences in terms of locoregional recurrences between concurrent or sequential radiohormonotherapy. Lung and skin fibroses due to concurrent treatment are still under debate. Nevertheless, late side effects appeared to be increased by such a treatment, particularly in hypersensitive patients identified at risk by the lymphocyte predictive test. Concurrent radiohormonotherapy should, thus, be delivered cautiously at least for these patients. This article details the potent advantages and risks of concurrent use of adjuvant hormonotherapy and radiotherapy in localized breast cancers.

Counselor motivational interviewing skills and young adult change talk articulation during brief motivational interventions.

The process of eliciting client language toward change (change talk [CT]) is implicated as a causal mechanism in motivational interviewing (MI) and brief motivational interventions (BMI). We investigated the articulation of counselor behaviors and CT during BMI with young men. We coded 149 sessions using the Motivational Interviewing Skill Code and summarized these codes into three counselor categories (MI-consistent [MICO], MI-inconsistent [MIIN], other) and three client categories (CT, counter CT [CCT], follow/neutral [F/N]). We then computed immediate transition frequencies and odds ratios using sequential analysis software. CT was significantly more likely following MICO behaviors, whereas MIIN behaviors only led to CCT and F/N. This strongly supports the use of MI skills to elicit CT during BMI with young men, whose speech also predicted counselor behaviors (particularly CT to MICO and CCT to MIIN). Additional analyses showed that among MICO behaviors, reflective listening may be a particularly powerful technique to elicit CT.

One minute of grief: Emotional processing in short-term dynamic psychotherapy for adjustment disorder.

OBJECTIVE: Depth of emotional processing has shown to be related to outcome across approaches to psychotherapy. Moreover, a specific emotional sequence has been postulated and tested in several studies on experiential psychotherapy (Pascual-Leone & Greenberg, 2007). This process-outcome study aims at reproducing the sequential model of emotional processing in psychodynamic psychotherapy for adjustment disorder and linking these variables with ultimate therapeutic outcome. METHOD: In this study, 32 patients underwent short-term dynamic psychotherapy. On the basis of reliable clinical change statistics, a subgroup (n = 16) presented with good outcome and another subgroup (n = 16) had a poor outcome in the end of treatment. The strongest alliance session of each case was rated using the observer-rated system Classification of Affective Meaning States. Reliability coefficients for the measure were excellent (κ = .82). RESULTS: Using 1 min as the fine-grained unit of analysis, results showed that the experience of fundamentally adaptive grief was more common in the in-session process of patients with good outcome, compared with those with poor outcomes (χ2 = 6.56, p = .01, d = 1.23). This variable alone predicted 19% of the change in depressive symptoms as measured by the Beck Depression Inventory at the end of treatment. Moreover, sequences of the original model were supported and related to outcome. CONCLUSIONS: These results are discussed within the framework of the sequential model of emotional processing and its possible relevance for psychodynamic psychotherapy. (PsycINFO Database Record (c) 2015 APA, all rights reserved).

A randomized, double blind, placebo-controlled trial of pioglitazone in combination with riluzole in amyotrophic lateral sclerosis.

BACKGROUND: Pioglitazone, an oral anti-diabetic that stimulates the PPAR-gamma transcription factor, increased survival of mice with amyotrophic lateral sclerosis (ALS). METHODS/PRINCIPAL FINDINGS: We performed a phase II, double blind, multicentre, placebo controlled trial of pioglitazone in ALS patients under riluzole. 219 patients were randomly assigned to receive 45 mg/day of pioglitazone or placebo (one: one allocation ratio). The primary endpoint was survival. Secondary endpoints included incidence of non-invasive ventilation and tracheotomy, and slopes of ALS-FRS, slow vital capacity, and quality of life as assessed using EUROQoL EQ-5D. The study was conducted under a two-stage group sequential test, allowing to stop for futility or superiority after interim analysis. Shortly after interim analysis, 30 patients under pioglitazone and 24 patients under placebo had died. The trial was stopped for futility; the hazard ratio for primary endpoint was 1.21 (95% CI: 0.71-2.07, p = 0.48). Secondary endpoints were not modified by pioglitazone treatment. Pioglitazone was well tolerated. CONCLUSION/SIGNIFICANCE: Pioglitazone has no beneficial effects on the survival of ALS patients as add-on therapy to riluzole. TRIAL REGISTRATION: Clinicaltrials.gov NCT00690118.

Oscillatory magnetic tweezers based on ferromagnetic beads and simple coaxial coils

We report the design and validation of simple magnetic tweezers for oscillating ferromagnetic beads in the piconewton and nanometer scales. The system is based on a single pair of coaxial coils operating in two sequential modes: permanent magnetization of the beads through a large and brief pulse of magnetic field and generation of magnetic gradients to produce uniaxial oscillatory forces. By using this two step method, the magnetic moment of the beads remains constant during measurements. Therefore, the applied force can be computed and varies linearly with the driving signal. No feedback control is required to produce well defined force oscillations over a wide bandwidth. The design of the coils was optimized to obtain high magnetic fields (280 mT) and gradients (2 T/m) with high homogeneity (5% variation) within the sample. The magnetic tweezers were implemented in an inverted optical microscope with a videomicroscopy-based multiparticle tracking system. The apparatus was validated with 4.5 ¿m magnetite beads obtaining forces up to ~2 pN and subnanometer resolution. The applicability of the device includes microrheology of biopolymer and cell cytoplasm, molecular mechanics, and mechanotransduction in living cells.

Towards a theory of the credit-risk balance sheet (II). The evolution of its structure

This article has an immediate predecessor, upon which it is based and with which readers must necessarily be familiar: Towards a Theory of the Credit-Risk Balance Sheet (Vallverdú, Somoza and Moya, 2006). The Balance Sheet is conceptualised on the basis of the duality of a credit-based transaction; it deals with its theoretical foundations, providing evidence of a causal credit-risk duality, that is, a true causal relationship; its characteristics, properties and its static and dynamic characteristics are analyzed. This article, which provides a logical continuation to the previous one, studies the evolution of the structure of the Credit-Risk Balance Sheet as a consequence of a business¿s dynamics in the credit area. Given the Credit-Risk Balance Sheet of a company at any given time, it attempts to estimate, by means of sequential analysis, its structural evolution, showing its usefulness in the management and control of credit and risk. To do this, it bases itself, with the necessary adaptations, on the by-now classic works of Palomba and Cutolo. The establishment of the corresponding transformation matrices allows one to move from an initial balance sheet structure to a final, future one, to understand its credit-risk situation trends, as well as to make possible its monitoring and control, basic elements in providing support for risk management.

Fast liver catabolism of C1q in patients with paraproteinaemia and depletion of the classical pathway of complement.

The main clinical features in four patients with IgG1k paraproteinaemia and acquired complement deficiency included xanthomatous skin lesions (in three), panniculitis (in three) and hepatitis (in two). Hypocomplementaemia concerned the early classical pathway components--in particular C1q. Metabolic studies employing 125I-C1q revealed a much faster catabolism of this protein in the four patients than in five normal controls and three patients with cryoglobulinaemia (mean fractional catabolic rates respectively: 23.35%/h; 1.44%/h; 5.84%/h). Various experiments were designed to characterize the mechanism of the hypocomplementaemia: the patients' serum, purified paraprotein, blood cells, bone marrow cells, or xanthomatous skin lesions did not produce significant complement activation or C1q binding. When three of the patients (two with panniculitis and hepatitis) were injected with 123I-C1q, sequential gamma-camera imaging demonstrated rapid accumulation of the radionuclide in the liver, suggesting that complement activation takes place in the liver where it could produce damage.

Control of jasmonate biosynthesis and senescence by miR319 targets.

Considerable progress has been made in identifying the targets of plant microRNAs, many of which regulate the stability or translation of mRNAs that encode transcription factors involved in development. In most cases, it is unknown, however, which immediate transcriptional targets mediate downstream effects of the microRNA-regulated transcription factors. We identified a new process controlled by the miR319-regulated clade of TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) transcription factor genes. In contrast to other miRNA targets, several of which modulate hormone responses, TCPs control biosynthesis of the hormone jasmonic acid. Furthermore, we demonstrate a previously unrecognized effect of TCPs on leaf senescence, a process in which jasmonic acid has been proposed to be a critical regulator. We propose that miR319-controlled TCP transcription factors coordinate two sequential processes in leaf development: leaf growth, which they negatively regulate, and leaf senescence, which they positively regulate.

White luminescence from Si+ and C+ ion-implanted SiO2 films

The microstructural and optical analysis of SiO2 layers emitting white luminescence is reported. These structures have been synthesized by sequential Si+ and C+ ion implantation and high-temperature annealing. Their white emission results from the presence of up to three bands in the photoluminescence (PL) spectra, covering the whole visible spectral range. The microstructural characterization reveals the presence of a complex multilayer structure: Si nanocrystals are only observed outside the main C-implanted peak region, with a lower density closer to the surface, being also smaller in size. This lack of uniformity in their density has been related to the inhibiting role of C in their growth dynamics. These nanocrystals are responsible for the band appearing in the red region of the PL spectrum. The analysis of the thermal evolution of the red PL band and its behavior after hydrogenation shows that carbon implantation also prevents the formation of well passivated Si/SiO2 interfaces. On the other hand, the PL bands appearing at higher energies show the existence of two different characteristics as a function of the implanted dose. For excess atomic concentrations below or equal to 10%, the spectra show a PL band in the blue region. At higher doses, two bands dominate the green¿blue spectral region. The evolution of these bands with the implanted dose and annealing time suggests that they are related to the formation of carbon-rich precipitates in the implanted region. Moreover, PL versus depth measurements provide a direct correlation of the green band with the carbon-implanted profile. These PL bands have been assigned to two distinct amorphous phases, with a composition close to elemental graphitic carbon or stoichiometric SiC.

Bipolar pulsed excitation of erbium-doped nanosilicon light emitting diodes

High quantum efficiency erbium doped silicon nanocluster (Si-NC:Er) light emitting diodes (LEDs) were grown by low-pressure chemical vapor deposition (LPCVD) in a complementary metal-oxide-semiconductor (CMOS) line. Erbium (Er) excitation mechanisms under direct current (DC) and bipolar pulsed electrical injection were studied in a broad range of excitation voltages and frequencies. Under DC excitation, Fowler-Nordheim tunneling of electrons is mediated by Er-related trap states and electroluminescence originates from impact excitation of Er ions. When the bipolar pulsed electrical injection is used, the electron transport and Er excitation mechanism change. Sequential injection of electrons and holes into silicon nanoclusters takes place and nonradiative energy transfer to Er ions is observed. This mechanism occurs in a range of lower driving voltages than those observed in DC and injection frequencies higher than the Er emission rate.