Beware of your Cre-Ation: lacZ expression impairs neuronal integrity and hippocampus-dependent memory.

Expression of the lacZ-sequence is a widely used reporter-tool to assess the transgenic and/or transfection efficacy of a target gene in mice. Once activated, lacZ is permanently expressed. However, protein accumulation is one of the hallmarks of neurodegenerative diseases. Furthermore, the protein product of the bacterial lacZ gene is ß-galactosidase, an analog to the mammalian senescence-associated ß-galactosidase, a molecular marker for aging. Therefore we studied the behavioral, structural and molecular consequences of lacZ expression in distinct neuronal sub-populations. lacZ expression in cortical glutamatergic neurons resulted in severe impairments in hippocampus-dependent memory accompanied by marked structural alterations throughout the CNS. In contrast, GFP expression or the expression of the ChR2/YFP fusion product in the same cell populations did not result in either cognitive or structural deficits. GABAergic lacZ expression caused significantly decreased hyper-arousal and mild cognitive deficits. Attenuated structural and behavioral consequences of lacZ expression could also be induced in adulthood, and lacZ transfection in neuronal cell cultures significantly decreased their viability. Our findings provide a strong caveat against the use of lacZ reporter mice for phenotyping studies and point to a particular sensitivity of the hippocampus formation to detrimental consequences of lacZ expression. © 2016 Wiley Periodicals, Inc.

FKBP51 inhibits GSK3ß and augments the effects of distinct psychotropic medications.

Psychotropic medications target glycogen synthase kinase 3ß (GSK3ß), but the functional integration with other factors relevant for drug efficacy is poorly understood. We discovered that the suggested psychiatric risk factor FK506 binding protein 51 (FKBP51) increases phosphorylation of GSK3ß at serine 9 (pGSK3ß(S9)). FKBP51 associates with GSK3ß mainly through its FK1 domain; furthermore, it also changes GSK3ß's heterocomplex assembly by associating with the phosphatase PP2A and the kinase cyclin-dependent kinase 5. FKBP51 acts through GSK3ß on the downstream targets Tau, ß-catenin and T-cell factor/lymphoid enhancing factor (TCF/LEF). Lithium and the antidepressant (AD) paroxetine (PAR) functionally synergize with FKBP51, as revealed by reporter gene and protein association analyses. Deletion of FKBP51 blunted the PAR- or lithium-induced increase in pGSK3ß(S9) in cells and mice and attenuated the behavioral effects of lithium treatment. Clinical improvement in depressive patients was predicted by baseline GSK3ß pathway activity and by pGSK3ß(S9) reactivity to ex vivo treatment of peripheral blood mononuclear lymphocytes with lithium or PAR. In sum, FKBP51-directed GSK3ß activity contributes to the action of psychotropic medications. Components of the FKBP51-GSK3ß pathway may be useful as biomarkers predicting AD response and as targets for the development of novel ADs.

Resonance-enhanced multiphoton ionization time-of-flight mass spectrometry for detection of nitrogen containing aliphatic and aromatic compounds: resonance-enhanced multiphoton ionization spectroscopic investigation and on-line analytical application.

Resonance-enhanced multiphoton ionization (REMPI) combined with time-of-flight mass spectrometry (TOFMS) is an analytical method capable of on-line monitoring of trace compounds in complex matrices. A necessary prerequisite for substance selective detection is spectroscopic investigation of the target molecules. Several organic nitrogen compounds comprising aliphatic and aromatic amines, nitrogen heterocyclic compounds, and aromatic nitriles are spectroscopically investigated with a tunable narrow bandwidth optical parametric oscillator (OPO) laser system providing a scannable wavelength range between 220 and 340 nm. These species are known as possible precursors in fuel-NO formation from combustion of solid fuels such as biomass and waste. A newly conceived double inlet system was used in this study, which allows rapid change between effusive and supersonic molecular beams. The resulting REMPI spectra of the compounds are discussed with respect to electronic transitions that could be utilized for a selective ionization of these compounds in complex mixtures such as combustion and process gases. The practicability of this approach is demonstrated by wavelength selected on-line REMPI-TOFMS detection of aniline and cyanonaphthalene in the burning chamber of a waste incineration plant. REMPI mass spectra recorded at different excitation wavelengths as well as variations in time show the utilization of species-selective REMPI-TOFMS detection for on-line monitoring of crucial substances in pollutant formation.

Investigation of NOx precursor compounds and other combustion by-products in the primary combustion zone of a waste-incineration plant using on-line, real-time mass spectrometry and Fourier-transform infrared spectrometry (FTIR).

On-line analysis of trace and bulk gas compounds in the burning chamber of a waste-incineration plant has been performed, with high temporal resolution, by use of a variety of distinctly different measurement techniques. Time-of-flight mass spectrometry was performed with simultaneous use of three ionization techniques-resonance-enhanced multiphoton ionization (REMPI), single-photon ionization (SPI), and electron-impact ionization (EI). Chemical-ionization mass spectrometry (CIMS), Fourier-transform infrared spectrometry (FTIR), and electrochemical methods were also used. Sampling was conducted by means of a newly developed air-cooled stainless steel lance, to cope with the high temperatures and elevated particle concentrations at the sampling location. Nitrogen species were mainly nitrogen monoxide, ammonia, and hydrogen cyanide (HCN), with a small amount (approximately 0.3%) of aromatic nitrogen compounds. NO, NH(3), and HCN are the main contributors to the NO(x)-formation process in the postulated fuel-NO reaction scheme dominant at this location. The NO recycling process thereby plays a major role. Changes in plant operating conditions have a noticeable impact only when the air supply is varied. For example, reduction of oxygen leads to an increase in the HCN fraction of the total nitrogen content and a decrease in the NO fraction, and vice versa.

On-line analysis of gas-phase composition in the combustion chamber and particle emission characteristics during combustion of wood and waste in a small batch reactor.

The emission of particulate matter and gaseous compounds during combustion of wood and refuse-derived fuel in a small batch reactor is investigated by laser mass-spectrometric on-line measurement techniques for gas-phase analysis and simultaneous registration of physical aerosol properties (number size distribution). The gas-phase composition is addressed by a laser-based mass spectrometric method, namely, vacuum-UV single-photon ionization time-of-flight mass spectrometry (VUV-SPI-TOFMS). Particle-size distributions are measured with a scanning mobility particle sizer. Furthermore, a photoelectric aerosol sensor is applied for detection of particle-bound polycyclic aromatic hydrocarbons. The different phases of wood combustion are distinguishable by both the chemical profiles of gas-phase components (e.g., polycyclic aromatic hydrocarbons, PAH) and the particle-size distribution. Furthermore, short disturbances of the combustion process due to air supply shortages are investigated regarding their effect on particle-size distribution and gas-phase composition, respectively. It is shown that the combustion conditions strongly influence the particle-size distribution as well as on the emission of particle-bound polycyclic aromatic hydrocarbons.

Comprehensive on-line characterization of complex gas mixtures by quasi-simultaneous resonance-enhanced multiphoton ionization, vacuum-UV single-photon ionization, and electron impact ionization in a time-of-flight mass spectrometer: setup and instrument characterization.

This paper reports on a newly developed mobile mass spectrometer for comprehensive on-line analysis of complex gas mixtures such as ambient air or industrial process gases. Three ionization methods, namely, the resonance-enhanced multiphoton ionization (REMPI), vacuum-ultraviolet single-photon ionization (SPI), and electron impact ionization (EI) are implemented in this instrument and can be operated (quasi-) simultaneously. By means of this setup, a wide range of compounds can be analyzed due to the unique ionization selectivitiy and sensitivity profiles provided by the different ionization techniques. The mass spectrometer is designed for field application even under severe conditions. The REMPI technique is suitable for the selective and soft ionization (without fragmentation) of aromatic compounds at trace level (ppbv/pptv). The also soft but less selective SPI technique with 118-nm vacuum-ultraviolet laser pulses is used as a second laser-based ionization method. Mass spectra obtained by this technique show profiles of most organic compounds (aliphatic and aromatic species) and of some low IP inorganic substances (e.g., ammonia, nitrogen oxide) down to ppbv concentrations. In addition to the laser-based ionization techniques, EI ionization can be used for analysis of the bulk components such as water, oxygen, nitrogen, and carbon dioxide as well as for detection of inorganic minor components such as HCN or HCl from combustion flue gases at ppmv concentration levels. Each method yields specific mass spectrometric information of the sample composition. Special techniques have been developed to combine the three ionization methods in a single mass spectrometer and to allow the quasi-parallel application of all three ionization techniques.

A capillary-based supersonic jet inlet system for resonance-enhanced laser ionization mass spectrometry: principle and first on-line process analytical applications.

A new supersonic jet inlet system for resonance-enhanced multiphoton ionization time-of-flight mass spectrometry (REMPI-TOFMS), based on a fused-silica capillary with an integral nozzle has been developed. The new jet inlet system generates a supersonic molecular beam that originates in the center of the ion source of the time-of-flight mass spectrometer. Because of the design of the inlet system, high spatial overlap of sample and laser beam (i.e., increased detection sensitivity) and excellent jet beam qualities are achieved with good adiabatic cooling properties of analyte molecules (i.e., considerably enhanced optical selectivity of the REMPI process). Furthermore, the inlet is very robust and chemically inert and contains no moving parts. As a result of these properties, the new inlet is perfectly suited for field applications of jet-REMPI. A first field application of a mobile supersonic jet-REMPI mass spectrometer equipped with the novel inlet technique is reported; namely, the concentration of monochlorobenzene, which is an indicator for the formation and emission of toxic polychlorinated dibenzo-p-dioxins/furans, PCDD/F) was measured on-line in the flue gas of a waste incineration plant.

Incidence and prognostic significance of fever following intracerebral hemorrhage.

OBJECTIVE: To investigate the incidence and prognostic significance of fever on presentation and during the subsequent 72 hours in patients with spontaneous supratentorial intracerebral hemorrhage (ICH). METHODS: We analyzed 251 patients. On admission, body temperature, Glasgow Coma Scale (GCS) score, age, sex, blood pressure, blood glucose level, and presumed origin of hemorrhage were analyzed. From the initial CT scan, hematoma volume, location, and presence of intraventricular hemorrhage were determined. From the first 72 hours, hematoma enlargement, duration of increased temperatures, blood pressure, and blood glucose level were determined. Outcome was classified on discharge with the Glasgow Outcome Scale (GOS) score. RESULTS: Outcomes included no symptoms in 23 (9%), moderate disability in 64 (26%), severe disability in 104 (41%), vegetative state in 5 (2%), and death in 55 (22%) patients. Prognostic factors retained from a logistic regression model with a dichotomized GOS scale (GOS score of 1 or 2 versus GOS score of 3 to 5) as response variables were GCS score of 7 or less, age older than 75 years, hematoma volume of more than 60 cm3, ventricular hemorrhage, and presence of a coagulation disorder (p < 0.05). Fever was associated with intraventricular hemorrhage. From 196 patients, data from the first 72 hours were analyzed. A total of 18 patients (9%) had normal temperatures throughout the study. The duration of fever (> or =37.5 degrees C) was less than 24 hours in 66 (34%), 24 to 48 hours in 70 (36%), and more than 48 hours in 42 patients (21%). Independent prognostic factors during the first 72 hours were duration of fever, secondary hemorrhage, GCS score of 7 or less, ventricular hemorrhage, hematoma volume of more than 60 cm3, duration of increased blood pressure of more than 48 hours, and duration of increased blood glucose of more than 48 hours. CONCLUSIONS: The incidence of fever after supratentorial ICH is high, especially in patients with ventricular hemorrhage. In patients surviving the first 72 hours after hospital admission, the duration of fever is associated with poor outcome and seems to be an independent prognostic factor in these patients.

Proventil HFA provides protection from exercise-induced bronchoconstriction comparable to proventil and ventolin.

INTRODUCTION: During the 1970s, scientists suggested that the growing use of chlorofluorocarbons (CFCs) was contributing to depletion of the stratospheric ozone layer with potentially harmful results. A committee on the ozone layer organized the preparation of the Montreal Protocol. This protocol mandated the cessation of production and use of CFCs by January 1, 1996. The primary exemption to this ban is for the use of CFCs as propellants in metered dose inhalers (MDIs) for the treatment of asthma. Suitable replacement hydrofluoroalkane (HFA) propellants, such as HFA-134a, for use in MDIs have been identified. Albuterol, a selective beta-adrenergic agonist, currently widely available for inhalation asthma therapy, has been reformulated in HFA-134a (Proventil HFA). OBJECTIVE; To compare the efficacy of Proventil HFA to Ventolin, Proventil, and placebo (HFA-134a) MDI in protecting asthmatic patients from exercise-induced bronchoconstriction. METHODS: This was a randomized, single-blind, placebo-controlled, 4-period crossover study of asthmatic patients with documented exercise-induced broncho-constriction. Twenty patients self administered two puffs of either Proventil HFA, Ventolin, Proventil or placebo, from an MDI, 30 minutes prior to performing a standardized exercise challenge at the study site. Spirometry was performed predose and 5, 10, 15, 30, 45, 60, 75, and 90 minutes after completion of the exercise challenge. Heart rate and blood pressure were measured just prior to spirometry and a 12-lead ECG was performed 15 minutes after completion of the exercise challenge for measurement of the QT corrected interval. RESULTS: The primary efficacy variable was the smallest percent change from the predose FEV1 following exercise. The smallest percent change from predose FEV1 for Proventil HFA was 2.0 +/- 9.9 SD, similar to the 2.0 +/- 11.4 SD for Ventolin, and the 3.6 +/- 10.2 SD for Proventil. The smallest percent change from predose FEV1 for each of the active treatments was significantly different from placebo, -23.7 +/- 14.5. Twelve of the patients had a > or = 20% fall in FEV1 post-exercise with placebo pretreatment, but only 1, 1, and 0 had > or = 20% FEV1 falls after treatment with Proventil HFA, Ventolin, and Proventil respectively. Changes in heart rate, blood pressure and QT corrected interval were similar for the three active treatments following exercise. CONCLUSIONS: Proventil HFA provides protection against exercise-induced bronchoconstriction comparable to Ventolin and Proventil and protection superior to placebo. Proventil HFA has a safety profile similar to Ventolin when used to prevent exercise-induced bronchoconstriction.

Treatment of cat allergy with T-cell reactive peptides.

We induced in allergic humans the counterpart of murine experimental T-cell tolerance. T-cell lines from cat-allergic humans were used to map T-cell epitopes for the principal allergen of cat dander, Fel d 1. Two peptides of 27 amino acids each were synthesized to contain the dominant epitopes (ALLERVAX CAT). After a safety trial, we carried out a blinded study of the dose required for efficacy. We randomly divided 95 cat-sensitive patients into placebo, 7.5 micrograms, 75 micrograms, and 750 micrograms groups. Patients received a subcutaneous injection weekly for 4 wk. Before and after treatment, patients were exposed in a room inhabited by live cats and scored by nose and lung symptoms. Baseline nasal and lung scores (+/-SEM) were 6.2 +/- 0.56 and 5.4 +/- 0.73 in the 750 micrograms group; 7.8 +/- 0.53 and 4.7 +/- 0.68 in the placebo group. Six weeks after treatment, scores adjusted for baseline differences were reduced in the 750 micrograms group: -2.3 +/- 4.9 and -2.3 +/- 0.59 compared with -0.84 +/- 0.50 and -0.85 +/- 0.62 in the placebo group. The 75 micrograms group showed intermediate effects and the 7.5 micrograms group no effect. Linear trend analysis indicated a significant dose response effect: p = 0.05 for nose and 0.03 for lung symptoms. Allergic side effects occurred an hour or more after the first 750 micrograms dose in 16 of 24 patients but required little or no treatment with one exception. T-cell reactive treatment peptides safely improved allergic responses to cats.

Factorial dose-response studies using frequency and magnitude of dose.

In the early stages of traditional drug development, the frequency of dosing (e.g., QD, BID, etc.) is typically determined by the pharmacokinetic properties of a compound. After an appropriate dose frequency is chosen, the magnitude of dose is then evaluated via parallel-group dose-response trials. For some drugs, however, blood levels at any given time may not be accurate predictors of clinical response, or the drug may not be absorbed systemically. In those instances, we propose the use of a factorial dose-response trial that simultaneously evaluates frequency of dosing and magnitude of dose. We consider this approach to selecting an appropriate dosing regimen to be more scientifically founded and more cost-effective, than independent evaluation of dose and frequency through separate clinical trials. Some design considerations and statistical analysis strategies for these factorial trials are presented in this paper.

Three-period crossover trial with ambulatory blood pressure monitoring for evaluating antihypertensive therapy.

A double-blind, three-period, crossover trial used 24-hour ambulatory blood pressure monitoring to compare diltiazem controlled diffusion (CD) 300 mg with placebo. Patients with hypertension (N = 43) were randomly assigned to one of four crossover treatment sequences of three treatment periods each. Ambulatory blood pressure was obtained at the end of each 4-week treatment period. Diltiazem CD significantly decreased diastolic and systolic blood pressure at bihourly ambulatory blood pressure evaluations (p < 0.05, all). However, when all ambulatory blood pressure monitoring data were combined into one statistical model, blood pressure reductions were quantifiably similar to those in the overall bihourly analysis, but with a consistent 24-hour antihypertensive effect for both diastolic and systolic blood pressure relative to that with placebo (i.e., parallel blood pressure profiles) and with increased precision. Mean +/- SE changes in diastolic and systolic blood pressure across the 24-hour dosing interval were -5.6 +/- 0.4 mm Hg and -7.6 +/- 0.5 mm Hg, respectively (p < 0.001, both). Therefore, by using a crossover design with ambulatory blood pressure monitoring, we showed diltiazem CD to reduce blood pressure consistently throughout a 24-hour dosing interval in comparison with placebo in patients with hypertension.

A dose-response trial of once-daily diltiazem.

This trial was performed to determine the safe and effective dosage range of once daily diltiazem (diltiazem CD) capsules for treatment of essential hypertension. Patients with essential hypertension having supine diastolic blood pressure values greater than or equal to 95 mm Hg and less than or equal to 110 mm Hg were randomly assigned to receive placebo or one of four doses of diltiazem CD: 90, 180, 360, or 540 mg. Blood pressure was measured at trough, 24 hours after the dose, and at the time of peak effect, 10 hours after the dose. Diltiazem CD lowered both supine diastolic and systolic blood pressure. A linear dose response was seen with changes in diastolic and systolic blood pressure and heart rate for trough and peak measurements. Trough/peak ratios for the 180, 360, and 540 mg doses were all greater than 0.50. Adverse effects were dose related; those most commonly reported were headache (8.6%), bradycardia (8.1%), and edema (7%), with bradycardia and edema possibly dose related. It is therefore concluded that diltiazem CD is a safe and effective antihypertensive agent.

On assessing interrater agreement for multiple attribute responses.

New methods are developed for assessing the extent of interrater agreement when each unit to be rated is characterized by a (possibly empty) subset of a specified set of distinct nominal attributes. For such multiple attribute response data, a two-rater concordance statistic is derived, and associated statistical inference-making procedures are provided. This concordance statistic is corrected for chance agreement by using an underlying hypergeometric model. Numerical examples are given to illustrate the proposed methodology, and comparisons to other agreement statistics (e.g., kappa) are made.

[Pollinosis in the area of the Dalmatian, Istrian and Croatian coast with special emphasis on Parietariae pollen]. / Polinoze na podrucju Dalmacije, Istre i Hrvatskog primorja s posebnim osvrtom na pelud Parietariae.

The distribution of pollen antigens of an area depends on geographic situation, climatic factors and vegetation. Geographic situation of examined area is in latitude from 42.5 degrees (Dubrovnik) to 45.5 degrees north (Rijeka), the climate is Mediterranean and botanically it is Eumediterranean area. In allergologic out-patient departments of Dubrovnik, Split, Sibenik, Zadar, Pula and Rijeka, 300 patients with pollinosis have been tested by the application of the prick method of group allergens of grass, tree and weed pollen, particularly of Parietariae (pellitory) pollen. The object of the investigation was to find out which pollen antigen is actual for the Adriatic area and distribution of Parietariae pollen, regarding the fact that it was almost the only cause of pollinosis in the south part of the Adriatic coast (Dubrovnik), Ninety healthy persons were also examined, 15 in each out-patient department, being the control. The results show that grass pollen is actual allergen in the north and middle Adriatic area, while in the south Adriatic it is without any importance. The number of people oversensitive to trees and weeds pollen is low. The oversensitiveness to Parietariae pollen appears all along the coast, its number decreasing from the south to the north. It is concluded that grass pollen is the main cause of pollinosis, in the area of the Croatian littoral and Istria, important in south and central Dalmatia and almost without importance in south Dalmatia. Parietariae pollen is actual allergen all along the coast, being almost the only cause of pollinosis in south Adriatic area.

Some analysis strategies for three-period changeover designs with two treatments.

Alternative analysis strategies for the three-period crossover design with two treatments are discussed in this paper. One analysis strategy involves a parametric model that incorporates the effects of interest. To implement this method, one usually assumes that the covariance structure for the data has the sphericity, or circularity, property. Alternative approaches that do not require this assumption are described. They are based on the parametric and non-parametric analysis of appropriate within-subject linear functions of the data. The advantage of these methods is that one only needs the assumption that the resulting linear functions for the respective subjects are independent and have a common distribution. The parametric approach also requires normality of the resulting within-subject linear functions for small sample situations. An extension of the non-parametric method is considered for cases in which the treatment sequences are randomly assigned within strata. The various methods are illustrated for a three-period crossover design involving two strata with randomly assigned treatment sequences of the form A:B:A and B:A:B.

Model calculations of the interaction of two parallel antiaromatic 4n pi-electron systems.

The nature of the interaction between decks of a pentalene dimer and an s-indacene dimer has been studied by semi-empirical MNDO/1 and MINDO/3 calculations for distances between decks of from 5 A to 2 A. In contradiction to qualitative predictions from a frontier orbital analysis, it is found that the 4n-4n pi-electron interaction between decks for such dimers is destabilizing for distances exceeding about 2.5 A.