Spontaneous Openings of NMDA Receptor Channels in Cultured Rat Hippocampal Neurons

Spontaneous and N-methyl-D-aspartate (NMDA)-evoked single-channel currents were studied in outside-out patches isolated from cultured rat hippocampal neurons. Both spontaneous and NMDA-evoked single-channel currents reversed at potentials close to 0 mV and exhibited multiple amplitude levels of similar amplitude. Both spontaneous and NMDA-evoked single-channel currents were inhibited by Mg²⁺ in a voltage-dependent manner and by 7-chlorokynurenic acid. The activity of spontaneous single-channel currents was reduced by the competitive NMDA receptor antagonists, but by one to three orders of magnitude less than expected assuming that the spontaneous activity is due to an ambient NMDA receptor agonist present in the extracellular solution. Our results suggest that, similar to other ligand-gated ion channels, NMDA receptor channels have a dual mode of activation - spontaneous and agonist induced.     

Cardiac CD68+ and stabilin-1+ macrophages in wound healing following myocardial infarction: From experiment to clinic

Myocardial infarction (MI) remains the leading cause of mortality and morbidity throughout the world. Macrophages are key innate immune cells that play a significant role in transition from the inflammatory to the regenerative phase during wound healing following MI. The scavenger receptor stabilin-1 is one of the most interesting macrophage biomarkers. This receptor contributes to wound healing, angiogenesis, and tissue remodeling. We suggested a research protocol using macrophage biomarkers to study the cellular basis of cardiac remodeling and healing in patients with acute MI. The purpose of the research was to translate experimental knowledge regarding macrophage subsets and their biomarkers in post-infarction myocardial regeneration into results observed in clinical settings. The study included 41 patients with fatal MI type 1. All patients were divided into four groups according to the timeline of MI histopathology. In addition to routine histopathological analysis, macrophage infiltration was assessed by immunohistochemistry. We used CD68 as a marker for the cells of the macrophage lineage and stabilin-1 as an M2-like macrophage biomarker. The number of CD68+ and stabilin-1+ macrophages in the infarct area increased and peaked in the regenerative phase and did not decrease in the late stage of MI. In the peri-infarct area, the number of CD68+ macrophages increased in the inflammatory phase, peaked during the reparative phase, and did not decrease in the late phase, while the number of stabilin-1+ macrophages increased in the regenerative phase and remained unchanged. Additionally, in the reparative phase, we observed increase in the number of CD68+ and stabilin-1+ macrophages in the non-infarct area. The research protocol suggested allowed us to translate experimental knowledge regarding macrophage subsets and their biomarkers in post-infarction myocardial regeneration into clinical data. Taken together, these results demonstrated biphasic cardiac macrophage response following acute MI somewhat similar to that in a murine model. The increase in stabilin-1+ macrophage infiltration noticed in the myocardium during the regenerative phase and the strong positive correlation between the number of these cells and timeline of MI histopathology enabled us to propose stabilin-1 as a diagnostic macrophage biomarker in myocardium wound healing in patients with acute MI.     

Bone mineral imaged in vivo by 31P solid state MRI of human wrists

Purpose:

To implement solid state ³¹P MRI (³¹P SMRI) in a clinical scanner to visualize bone mineral.

Materials and Methods:

Wrists of seven healthy volunteers were scanned. A quadrature wrist ³¹P transmit/receive coil provided strong B₁ and good signal‐to‐noise ratio (SNR). A ¹H‐³¹P frequency converter was constructed to enable detection of the ³¹P signal by means of the ¹H channel. Data points lost in the receiver dead time were recovered by a second acquisition with longer dwell time and lower gradient strength.

Results:

Three‐dimensional ³¹P images, showing only bone mineral of the wrist, were obtained with a clinical 3 Tesla (T) scanner. In the best overall case an image with isotropic resolution of ～5.1 mm and SNR of 30 was obtained in 37 min. ³¹P NMR properties (resonance line width 2 kHz and T₁ 17–19 s) of in vivo human bone mineral were measured.

Conclusion:

In vivo ³¹P SMRI visualization of human wrist bone mineral with a clinical MR scanner is feasible with suitable modifications to circumvent the scanners' limitations in reception of short‐T₂ signals. Frequency conversion methodology is useful for implementing ³¹P SMRI measurements on scanners which do not have multinuclear capability or for which the multinuclear receiver dead time is excessive. J. Magn. Reson. Imaging 2011;. © 2011 Wiley‐Liss, Inc.     

Hyperphosphorylated truncated protein tau induces caspase-3 independent apoptosis-like pathway in the Alzheimer's disease cellular model

Neurofibrillary degeneration and neuronal loss represent key pathological hallmarks of Alzheimer's disease (AD). It has been demonstrated that the decrease of total neuronal numbers correlates with the presence of neurofibrillary degeneration in AD brain. In order to unravel the mechanism leading to the cell death in AD, we developed a stably transfected human neuroblastoma cellular model with doxycycline-regulatable expression of AD truncated tau protein (AT tau, 151-391 4R). Cells expressing the longest tau isoform (Tau 40) were used as a control. We found that more than 80% of the total amount of AT tau and Tau 40 were phosphorylated. Strikingly, both AT tau and Tau 40 reduced the metabolic activity of the cells in a time-dependent manner (p < 0.0001) suggesting that tau overexpression slows down cell proliferation. However, AT tau showed significantly higher toxicity than Tau 40 (p < 0.0001), which indicates that truncation leads to a toxic gain of function. The analysis of the type of the cell death revealed the characteristic features of apoptosis such as cell shrinkage, nuclear, and DNA fragmentation. However, we did not find either the activation of executive caspase (caspase-3) or the caspase cleavage products (PARP and fodrin). These results show that posttranslationally modified truncated tau protein induces caspase-3-independent apoptosis-like programmed cell death, a phenomenon we term tauoptosis.     

Regulatory assessment of in vitro skin corrosion and irritation data within the European framework: Workshop recommendations

Validated in vitro methods for skin corrosion and irritation were adopted by the OECD and by the European Union during the last decade. In the EU, Switzerland and countries adopting the EU legislation, these assays may allow the full replacement of animal testing for identifying and classifying compounds as skin corrosives, skin irritants, and non irritants. In order to develop harmonised recommendations on the use of in vitro data for regulatory assessment purposes within the European framework, a workshop was organized by the Swiss Federal Office of Public Health together with ECVAM and the BfR. It comprised stakeholders from various European countries involved in the process from in vitro testing to the regulatory assessment of in vitro data. Discussions addressed the following questions: (1) the information requirements considered useful for regulatory assessment; (2) the applicability of in vitro skin corrosion data to assign the corrosive subcategories as implemented by the EU Classification, Labelling and Packaging Regulation; (3) the applicability of testing strategies for determining skin corrosion and irritation hazards; and (4) the applicability of the adopted in vitro assays to test mixtures, preparations and dilutions. Overall, a number of agreements and recommendations were achieved in order to clarify and facilitate the assessment and use of in vitro data from regulatory accepted methods, and ultimately help regulators and scientists facing with the new in vitro approaches to evaluate skin irritation and corrosion hazards and risks without animal data.     

New Chemically Resistant Coating Systems with Progressive Incorporation of Hazardous Waste in Polyurethane and Epoxy Matrices

New types of highly chemically resistant coating systems, primarily intended for concrete and metal substrates, were designed and experimentally verified in the paper. Secondary raw materials in optimal amounts, including solidified hazardous waste (e.g., end product and cement bypass dust), were used as microfillers. The polymer coating systems, containing pre-treated hazardous waste (HW), showed high abrasion resistance and excellent adhesion to metal and concrete surfaces. Based on polyurethane and epoxy resins, the coatings can be used in environments where aggressive chemical media act, such as sewers and the chemical industry. The developed polymeric coating systems showed even better properties than the compared reference coating systems. The chemical resistance of the three-layer coating systems was evaluated both visually and based on changes in mechanical properties, such as hardness and adhesion. The microstructure of the coating systems was also monitored using a digital optical microscope and a scanning electron microscope with energy dispersive X-ray analysis (SEM-EDX) after chemical stress. It was observed that the particles of HW were fully incorporated into the polymer matrix of the coating systems.     

Crack-Resistant Cements under Drying: Results from Ring Shrinkage Tests and Multi-Physical Modeling

Cementitious materials exhibit shrinkage strain on drying, leading easily to crack formation when internally or externally restrained. It is known that cements with a slow strength gain show higher crack resistance under external drying. The ring shrinkage test can be considered an accelerated method for cracking tendency due to existing historical correlations between ring cracking time and long-term surface concrete cracking. The experimental campaign used ring shrinkage tests on 25 mortars, covering 10 commercial cements and 15 cements produced on demand, covering Portland cements and blended cements up to a 30% slag substitution. The results show that the restrained ring cracking time generally increases with lower Blaine fineness and higher slag substitution in 6 to over 207 days’ span. Upper limits for crack-resistant cements were proposed for 2-day compressive strength and Blaine fineness, in the case of Portland cements: 27.7 MPa and 290 m²/kg, respectively. A hygro-mechanical model successfully replicated strain evolution with crack formation and brittle failure. Only two out of ten commercial cements were classified as crack-resistant, while the ratio increased to 10 out of 15 cements which were produced on demand.     

A Study of Physicochemical Properties of Stockpile and Ponded Coal Ash

The article describes chemical and also selected physical properties of ponded high temperature fly ash (FA) and bottom ash (BA) from a Mělník lignite power plant located in the Czech Republic. The research was carried out on samples obtained from drills with a depth of up to 54 m and the age of the samples retrieved from the lowest layers of the stockpile dating back to 1960. At the same time, a comparison was made with fresh fly ash and fresh bottom ash obtained from the identical power plant. The study analyzed a total of 98 stockpile samples. The properties selected were studied across the entire stockpile, namely moisture content, specific density, specific surface, carbon content, elemental and phase composition, pH, electrical conductivity and leachability. SEM analyses were also performed. The performed measurements of chemical properties proved the chemical stability of the material even after several decades of storage in the stockpile. The largest changes are evident in the results of the analyses related to the leachability of SO₃, Cl⁻ and F⁻. In contrast, the pH does not change significantly, and the composition is pH neutral or alkaline. Regarding ponded BA, particle disintegration was noted depending on the increasing core borehole depth.     

Lack of effect of boar seminal vesicle fluid immunosuppressor factor on embryo development

The effects of an immunosuppressive factor (ISF) isolated from boar seminal vesicle fluid on in vitro and in vivo mouse development were investigated. It was found that the zona pellucida of porcine and mouse oocytes pre-incubated with ISF reacted in indirect immunofluorescence with antibodies against ISF. Further results indicated that the boar ISF had no effect on embryo development.