Superior antitumoral activity of dimerized targeted single-chain TRAIL fusion proteins under retention of tumor selectivity.

Although targeting of the death receptors (DRs) DR4 and DR5 still appears a suitable antitumoral strategy, the limited clinical responses to recombinant soluble TNF-related apoptosis inducing ligand (TRAIL) necessitate novel reagents with improved apoptotic activity/tumor selectivity. Apoptosis induction by a single-chain TRAIL (scTRAIL) molecule could be enhanced >10-fold by generation of epidermal growth factor receptor (EGFR)-specific scFv-scTRAIL fusion proteins. By forcing dimerization of scFv-scTRAIL based on scFv linker modification, we obtained a targeted scTRAIL composed predominantly of dimers (Db-scTRAIL), exceeding the activity of nontargeted scTRAIL ∼100-fold on Huh-7 hepatocellular and Colo205 colon carcinoma cells. Increased activity of Db-scTRAIL was also demonstrated on target-negative cells, suggesting that, in addition to targeting, oligomerization equivalent to an at least dimeric assembly of standard TRAIL per se enhances apoptosis signaling. In the presence of apoptosis sensitizers, such as the proteasomal inhibitor bortezomib, Db-scTRAIL was effective at picomolar concentrations in vitro (EC(50) ∼2 × 10(-12) M). Importantly, in vivo, Db-scTRAIL was well tolerated and displayed superior antitumoral activity in mouse xenograft (Colo205) tumor models. Our results show that both targeting and controlled dimerization of scTRAIL fusion proteins provides a strategy to enforce apoptosis induction, together with retained tumor selectivity and good in vivo tolerance.

[Evidence-based pharmacotherapy and other somatic treatment approaches for obsessive-compulsive disorder: state of the art]. / Evidenzbasierte Pharmakotherapie und andere somatische Therapieverfahren bei Zwangsstörungen : State of the art.

Meta-analyses of the randomized controlled trials (RCT) in obsessive-compulsive disorder (OCD) have clearly demonstrated that selective serotonin reuptake inhibitors (SSRIs) are the medication treatment of choice, while cognitive behavioural therapy (CBT) with exposure and response prevention is the psychotherapy of choice in OCD. Several guidelines emphasized that SSRIs are the first choice of medication in OCD. It has been noted that these agents may need to be given at a higher dose, and for a longer duration, than is usually the case in disorders such as depression. In the management of refractory patients, medication history should be carefully reviewed and adherence to the recommendations of the guideline established. Antipsychotics (risperidone, quetiapine, haloperidol) are currently the pharmacotherapy augmentation strategy of choice. In those OCD patients who fail to respond to a range of SSRIs and augmentation strategies combined with CBT, more unusual interventions (including deep brain stimulation) can be considered.

Obsessive-Compulsive Disorder (OCD) is still an unrecognised disorder: a study on the recognition of OCD in psychiatric outpatients.

OBJECTIVE: The study aims to investigate the recognition of patients with Obsessive-Compulsive Disorder (OCD) in psychiatric outpatients. SUBJECTS AND METHODS: A total of 2282 outpatients seen at 14 different psychiatric clinics in South Germany were asked to participate in the study. Five hundred and eighty-nine (30%) of the original 2282 patients met screening criteria for OCD, and of those, 237 (42%) participated in the final interview including DSM-IV diagnosis, and comorbidity. RESULTS: Sixty-nine of 589 participating patients who screened positively for obsessive symptoms actually had an Obsessive-Compulsive Disorder. Only 19 (28%) of the outpatients diagnosed with OCD according to DSM-IV criteria were also given this diagnosis by their consultant. The psychopathology scores indicated that the OCD patients had clinically relevant OCD with a mean Yale-Brown Obsessive Compulsive Score (Y-BOCS) of 17.5 (± 5.4), and a mean Clinical Global Impression Score (CGI) of 5.2 (± 1.2). CONCLUSIONS: In outpatient clinics over 70% of OCD patients remain unrecognised and thus untreated by consultants. Screening questions provide a rapid way of identifying those who may have OCD and should be incorporated into every mental state examination by consultants.

In situ ATR-FTIR characterization of primary cement interfaces of the barnacle Balanus amphitrite.

A method is presented for characterizing primary cement interfaces of barnacles using in situ attenuated total reflection-Fourier transform infrared spectroscopy. Primary cement of the barnacle, Balanus amphitrite (Amphibalanus amphitrite), was characterized without any disruption to the original cement interface, after settling and growing barnacles directly on double sided polished germanium wafers. High-quality IR spectra were acquired of live barnacle cement interfaces, providing a spectroscopic fingerprint of cured primary cement in vivo with the barnacle adhered to the substratum. Additional spectra were also acquired of intact cement interfaces for which the upper portion of the barnacle had been removed leaving only the base plate and cement layer attached to the substratum. This allowed further characterization of primary cement interfaces that were dried or placed in D(2)O. The resulting spectra were consistent with the cement being proteinaceous, and allowed analysis of the protein secondary structure and water content in the cement layer. The estimated secondary structure composition was primarily beta-sheet, with additional alpha-helix, turn and unordered components. The cement of live barnacles, freshly removed from seawater, was estimated to have a water content of 20-50% by weight. These results provide new insights into the chemical properties of the undisturbed barnacle adhesive interface.

Evaluation of sampling tools for environmental sampling of bacterial endospores from porous and nonporous surfaces.

AIMS: Having and executing a well-defined and validated sampling protocol is critical following a purposeful release of a biological agent for response and recovery activities, for clinical and epidemiological analysis and for forensic purposes. The objective of this study was to address the need for validated sampling and analysis methods called out by the General Accounting Office and others to systematically compare the collection efficiency of various swabs and wipes for collection of bacterial endospores from five different surfaces, both porous and nonporous. This study was also designed to test the collection and extraction solutions used for endospore recovery from swabs and wipes. METHODS AND RESULTS: Eight collection tools, five swabs and three wipes, were used. Three collection/preservation solutions were evaluated: an ink jet aerosol generator was used to apply Bacillus subtilis endospores to five porous and nonporous surfaces. The collection efficiencies of the swabs and wipes were compared using a statistical multiple comparison analysis. CONCLUSIONS: The ScottPure wipe had the highest collection efficiency and phosphate-buffered saline (PBST) with 0.3% Tween was the best collection solution of those tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Validated sampling for potential biological warfare is of significant importance and this study answered some relevant questions.

Enhanced perceived responsibility decreases metamemory but not memory accuracy in obsessive-compulsive disorder (OCD).

Mixed findings have been obtained in prior research with respect to the presence and severity of memory and metamemory deficits in obsessive-compulsive disorder (OCD). We tested the hypothesis that experimentally induced increments of subjective responsibility would lead to a disproportionately strong decline of memory confidence and enhanced response latencies in OCD while leaving memory accuracy unaffected. Twenty-eight OCD patients and 28 healthy controls were presented a computerized memory test framed with two different scenarios. In the neutral scenario, the participant was requested to imagine purchasing 15 items from a do-it-yourself store. In the recognition phase, the 15 needed items were presented along with 15 distractor items. The participant was asked to decide whether items were on his or her shopping list or not, graded by subjective confidence. In the responsibility scenario, the general experimental setup was analogous except that the participant now had to envision that he or she was a helper in a region recently struck by an earthquake, dispatched to provide 15 urgently needed goods from a nearby town. In line with prior work by our group, samples did not differ in either condition on memory accuracy in a subsequent recognition task. As hypothesized, OCD participants were less certain in their responses for the high responsibility condition than controls. Whereas patients and controls did not differ in their subjective estimates for memorized items, patients expressed stronger doubt that their earthquake mission was successful. The findings indicate that low memory confidence in OCD may only be elicited in situations where perceived responsibility is high and that patients may share higher performance standards ("good is not good enough") than controls when perceived responsibility is inflated.

A new understanding of attention-deficit disorders--beyond the age-at-onset criterion of DSM-IV.

For decades, ADHD has been seen primarily as a behavior disorder affecting only young children, primarily boys, causing them to be inattentive and disruptive in school. Research has now established that ADHD affects females as well as males, and that impairing symptoms, for most, persist into adulthood. It has also been shown that this disorder impacts many aspects of life that extend well beyond problems in school. This disorder impairs not only one's ability to sit still and listen, but also the capacity to organize tasks and materials, to sustain effort on tasks, and to utilize short-term memory for daily activities. Studies of children, adolescents, and adults with ADHD indicate that many suffer from a variety of cognitive impairments that extend beyond symptoms listed in the DSM-IV diagnostic criteria for ADHD. These include chronic problems in regulating alertness, in reading comprehension, and in modulating emotions, that occur more frequently in persons with ADHD than in the general population. This article describes recent research findings demonstrating the wide range of cognitive impairments associated with ADHD, which are related to a new model of ADHD, such as developmental impairment of executive functions of the brain.

Oscillating adhesive contacts between micron-scale tips and compliant polymers.

Adhesion of micron-scale probes with model poly(dimethylsiloxane), PDMS, elastomers was studied with a depth-sensing nanoindenter under oscillatory loading conditions. For contacts between diamond indenters (radius R = 5 or 10 microm) and PDMS, force-displacement curves were highly reversible and consistent with Johnson-Kendall-Roberts (JKR) behavior. However, our experiments have revealed striking differences between the experimental measurements of tip-sample interaction stiffness and the theoretical JKR stiffness. The measured stiffness was always greater than zero, even in the reflex portion of the curve (between the maximum adhesive force and release), where the JKR stiffness is negative. This apparent paradox can be resolved by considering the effects of viscoelasticity of PDMS on an oscillating crack tip in a JKR contact. Under well described conditions determined by oscillation frequency, sample viscoelastic properties, and the Tabor parameter (with variables R, reduced elastic modulus, E*, and interfacial energy, deltagamma), an oscillating crack tip will neither advance nor recede. In that case, the contact size is fixed (like that of a flat punch) at any given point on the load-displacement cycle, and the experimentally measured stiffness is equal to the equivalent punch stiffness. For a fixed oscillation frequency, a transition between JKR and punch stiffness can be brought about by an increase in radius of the probe or a decrease in PDMS modulus. Additionally, varying the oscillation frequency for a fixed E*, R, and deltagamma also resulted in transition between JKR and punch stiffness in a predictable manner. Comparisons of experiments and theory for an oscillating viscoelastic JKR contact are presented. The storage modulus and surface energy from nanoscale JKR stiffness measurements were compared to calculated values and those measured with conventional nanoindentation and JKR force-displacement analyses.

Neutralizing increases discomfort associated with obsessional thoughts: an experimental study with obsessional patients.

Cognitive-behavioral theories suggest that the development of neutralizing is crucial in the development and persistence of obsessional problems (OCD). Twenty-nine patients with a Diagnostic and Statistical Manual of Mental Disorders (4th ed., American Psychiatric Association, 1994) diagnosis of OCD were randomly allocated to 2 conditions. Both listened to repeated recorded presentations of their intrusive thoughts and either neutralized (experimental group) or distracted themselves (control). Discomfort was rated during this 1st phase and then during a 2nd phase without neutralizing or distraction. The experimental group showed a similar level of discomfort in the 1st phase, which significantly reduced during the period compared with controls. The experimental group experienced significantly more discomfort during the 2nd phase, and significantly stronger urges to neutralize and distract at the end of this phase than controls.

Cellular profile of cytokine production in a patient with visceral leishmaniasis: gammadelta+ T cells express both type 1 cytokines and interleukin-10.

The cytokine profile of CD4+, CD8+ T cells, gammadelta+ T cells and natural killer (NK) cells (CD94+CD3-) was studied in a patient with visceral leishmaniasis (VL). The otherwise healthy, human immunodeficiency virus-negative patient acquired the disease in Tuscany, Italy. Diagnosis was made by demonstration of high concentrations of antibodies against Leishmania antigens in serum. Flow cytometry for the detection of intracellular interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-6, IL-10, IL-13 and tumour necrosis factor (TNF)-alpha expression in peripheral blood mononuclear cells stimulated with phorbol 12-myristate 13-acetate and ionomycin was performed, followed by treatment with liposomal amphotericin B. CD4+ cells were identified as major cytokine-expressing cells, capable of producing both type 1 and type 2 cytokines. A high frequency of IL-4- and IL-13-expressing CD8+ cells was noted. NK cells and gammadelta+ T cells, thought to be involved in innate host defences against Leishmania, expressed IFN-gamma and TNF-alpha. Ten per cent of gammadelta+ T cells expressed IL-10, predominantly together with IFN-gamma, suggesting additional immune-regulatory roles for this T-cell subset in VL.

Immunological tolerance to inhaled antigen.

Regulatory mechanisms exist in the immune system to limit the induction of pathogenic responses to antigens encountered within the respiratory tract. The development of allergic disease is thought to arise as a result of the breakdown in these regulatory processes. In this review we examine the nature of immune responses generated to inhaled protein antigens and the mechanisms used to establish tolerance to inhaled antigens.

An algorithm for automated bacterial identification using matrix-assisted laser desorption/ionization mass spectrometry.

An algorithm for bacterial identification using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is being developed. This mass spectral fingerprint comparison algorithm is fully automated and statistically based, providing objective analysis of samples to be identified. Based on extraction of reference fingerprint ions from test spectra, this approach should lend itself well to real-world applications where samples are likely to be impure. This algorithm is illustrated using a blind study. In the study, MALDI-MS fingerprints for Bacillus atrophaeus ATCC 49337, Bacillus cereus ATCC 14579T, Escherichia coli ATCC 33694, Pantoea agglomerans ATCC 33243, and Pseudomonas putida F1 are collected and form a reference library. The identification of test samples containing one or more reference bacteria, potentially mixed with one species not in the library (Shewanella alga BrY), is performed by comparison to the reference library with a calculated degree of association. Out of 60 samples, no false positives are present, and the correct identification rate is 75%. Missed identifications are largely due to a weak B. cereus signal in the bacterial mixtures. Potential modifications to the algorithm are presented and result in a higher than 90% correct identification rate for the blind study data, suggesting that this approach has the potential for reliable and accurate automated data analysis of MALDI-MS.

Use of an internal control for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of bacteria.

A method to aid in the analysis of bacterial samples of unknown concentration by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is demonstrated. It is shown that in MALDI analysis of bacteria, the intensities of resulting peaks in spectra are sensitive to the microbial concentration. At the high and low ends of the concentration range, no signal can be obtained, leaving very concentrated or very dilute samples indistinguishable. The addition of cytochrome c as an internal control allows the differentiation of these concentrated and dilute samples. The presence of the internal control causes only a 20% to 30% decrease in signal intensity when the bacterial concentration is optimum. However, the signal quality is improved when the internal control is added to some low concentrations of bacteria.

Reproducibility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for replicate bacterial culture analysis.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to demonstrate the reproducibility of bacterial spectra collected on different days. The reproducibility of analysis by MALDI-MS of intact Escherichia coli and Bacillus atrophaeus is presented as a replicate culture study in which spectra were collected on ten different occasions over a three-month period and by two different operators. The analysis resulted in the detection of specific biomarkers in the m/z 2000-20 000 range. Some of the peaks in the Escherichia coli spectra are identified by comparison with other published work. All of the spectra obtained are reproducible over the course of the experiment, but operator variability does exist. The Escherichia coli spectra show operator variability while the Bacillus atrophaeus spectra do not. This work demonstrates the utility of MALDI in obtaining consistent spectra from bacteria over a period of time.

Extracting and visualizing matrix-assisted laser desorption/ionization time-of-flight mass spectral fingerprints.

We have developed a method for constructing and extracting matrix-assisted laser desorption/ionization (MALDI) fingerprints. This method is fully automated and statistically based, allowing a large number of spectra to be analyzed at a time in an objective manner. This method can be used to extract the fingerprint of a particular analyte from a spectrum containing multiple analytes. Therefore, this method lends itself well to real-world applications where samples to be analyzed are likely to be impure. We illustrate this method on experimental results from a series of studies of E. coli and B. atrophaeus MALDI time-of-flight mass spectrometry (TOFMS) fingerprints.

Mental rotation, memory scanning, and the central bottleneck.

Two reaction-time experiments using the psychological refractory period paradigm examined whether two prominent tasks, i.e., mental rotation and memory scanning, require access to a single-channel mechanism and must therefore be performed sequentially with other operations requiring the same mechanism. On each trial, subjects made speeded responses to a tone (Exp. 1) or a character (Exp. 2, with symbolic SR-compatibility of the character manipulated) as Task 1 and to a letter (for blocks with mental rotation) or a digit (for blocks with memory scanning) as Task 2. The set-size effect was constant across SOAs, suggesting that memory scanning cannot be performed in parallel with response selection of Task 1. The effect of orientation, however, decreased with decreasing SOA. The decrease was even intensified if Task 1 bottleneck processes were prolonged by symbolic SR-compatibility. The exact pattern of underadditivity, however, was not predicted by current theories of dual-task performance. The results contradict a central bottleneck model but are in line with extensions of the model proposed by Meyer and Kieras.

Observation of noncovalent complexes to the avidin tetramer by electrospray ionization mass spectrometry.

Intact avidin-biotin and avidin-biotin maleimide noncovalent complexes have been observed by electrospray ionization mass spectrometry (ESI-MS) by using an extended mass range quadrupole mass spectrometer. By utilizing mild ES1 interface conditions, the expected solution behavior of four biotin or biotin maleimide molecules noncovalently binding to each avidin tetramer can be preserved in the gas phase. The ESI-MS results show the appropriate mass additions of 973 ± 60 Da for biotin and 1802 ± 40 Da for biotin maleimide to the avidin tetramer species. These results support the hypothesis that substantial retention of higher order structure is possible in the gas phase by using gentle ESI conditions.

New developments in microscale separations and mass spectrometry for biomonitoring: capillary electrophoresis and electrospray ionization mass spectrometry.

In this article, we briefly highlight the use of capillary electrophoresis for sampling, manipulating, and separating extremely small sample sizes. The extraordinary sensitivity that can be obtained by combined capillary electrophoresis-mass spectrometry is then demonstrated using recent results. We briefly describe the ability to detect noncovalently associated complexes (e.g., double-stranded DNA) by electrospray ionization-mass spectrometry, and conclude with recent results that show the potential for using high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry for characterization of biomolecules.

Collisionally activated dissociation and tandem mass spectrometry of intact hemoglobin beta-chain variant proteins with electrospray ionization.

Electrospray ionization collisionally activated dissociation (CAD) mass spectra of multiply charged human hemoglobin beta-chain variant proteins (146 amino acid residues, 15.9 kDa), generated in the atmospheric pressure/vacuum interface and in the collision quadrupole of a triple-quadrupole mass spectrometer, are shown and compared. Several series of structurally informative singly and multiply charged b- and y-mode product ions are observed, with cleavage of the Thr 50-Pro 51 CO-NH bond to produce the complementary y96 and b50 sequence ions as the most favored fragmentation pathway. The eight different beta-globin variants studied differ by a single amino acid substitution and can be differentiated from the observed m/z shifts of the assigned product ions. The overall fragmentation patterns for the variant polypeptides are very similar, with the exception of the Willamette form, in which Arg is substituted for Pro- 51, and multiply charged y96 product ions are not observed. Circular dichroism spectra of normal beta A and beta Willamette show very little difference under a variety of solvent conditions, indicating that fragmentation differences in their respective CAD mass spectra are substantially governed by primary rather than secondary structure.