Temperature regulates NF-κB dynamics and function through timing of A20 transcription.

NF-κB signaling plays a pivotal role in control of the inflammatory response. We investigated how the dynamics and function of NF-κB were affected by temperature within the mammalian physiological range (34 °C to 40 °C). An increase in temperature led to an increase in NF-κB nuclear/cytoplasmic oscillation frequency following Tumor Necrosis Factor alpha (TNFα) stimulation. Mathematical modeling suggested that this temperature sensitivity might be due to an A20-dependent mechanism, and A20 silencing removed the sensitivity to increased temperature. The timing of the early response of a key set of NF-κB target genes showed strong temperature dependence. The cytokine-induced expression of many (but not all) later genes was insensitive to temperature change (suggesting that they might be functionally temperature-compensated). Moreover, a set of temperature- and TNFα-regulated genes were implicated in NF-κB cross-talk with key cell-fate-controlling pathways. In conclusion, NF-κB dynamics and target gene expression are modulated by temperature and can accurately transmit multidimensional information to control inflammation.

Quantitative dynamic imaging of immune cell signalling using lentiviral gene transfer.

Live-cell imaging of fluorescent fusion proteins has transformed our understanding of mammalian cell signalling and function. However, some cellular systems such as immune cells are unsuitable or refractory to many existing transgene delivery methods thus limiting systematic analyses. Here, a flexible lentiviral gene transfer platform for dynamic time-lapse imaging has been developed and validated with single-molecule spectroscopy, mathematical modelling and transcriptomics and used for analysis of a set of inflammation-related signalling networks. Time-lapse imaging of nuclear factor kappa B (NF-κB), signal transducer and activator of transcription (STATs) and nuclear factor of activated T-cells (NFAT) in mammalian immune cell lines provided evidence for heterogeneous temporal encoding of inflammatory signals. In particular, the absolute quantification of single-cell responses over time via fluorescent correlation spectroscopy (FCS) showed that NF-κB p65 activation in response to tumour necrosis factor α (TNFα) was differentially encoded in variable amplitude of nuclear translocation between immune and non-immune cells. The absolute number of activated molecules was dictated in part by the cell size, suggesting a morphology-dependent regulatory mechanism. The developed platform will enable further absolute quantitative analyses of the dynamic interactions between signalling networks, in and between individual cells, allowing better integration with mathematical models of signalling networks.

Convergent evolution to an aptamer observed in small populations on DNA microarrays.

The development of aptamers on custom synthesized DNA microarrays, which has been demonstrated in recent publications, can facilitate detailed analyses of sequence and fitness relationships. Here we use the technique to observe the paths taken through sequence-fitness space by three different evolutionary regimes: asexual reproduction, recombination and model-based evolution. The different evolutionary runs are made on the same array chip in triplicate, each one starting from a small population initialized independently at random. When evolving to a common target protein, glucose-6-phosphate dehydrogenase (G6PD), these nine distinct evolutionary runs are observed to develop aptamers with high affinity and to converge on the same motif not present in any of the starting populations. Regime specific differences in the evolutions, such as speed of convergence, could also be observed.

Multiresidue method for the triphenylmethane dyes in fish: Malachite green, crystal (gentian) violet, and brilliant green.

Liquid chromatographic methods are presented for the quantitative and confirmatory determination of crystal violet (CV; also known as gentian violet), leucocrystal violet (LCV), brilliant green (BG), and leucobrilliant green (LBG) in catfish. LCV and LBG were oxidized to the chromic CV and BG by reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and residues were measured as the combined CV+/-LCV and BG+/-LBG. These methods are extensions of published methods for malachite green (MG) analysis to allow simultaneous determination of MG, CV, and BG. Residues were extracted from muscle with ammonium acetate buffer and acetonitrile, and extracts cleaned up using dichloromethane partitioning and solid-phase extraction. Extracts were analyzed by liquid chromatography with visible detection (LC-VIS). The method was validated for catfish fortified with LCV over the range 0.25-10 ngg(-1) and CV at 2 ngg(-1). Average recoveries were 90.6% (+/-8.1% R.S.D., n=45) for LCV and 84.4% (+/-4.2% R.S.D., n=6) for CV. The average recovery for samples fortified with BG or LBG over the range 0.5-10 ngg(-1) was 67.2% (+/-14.8% R.S.D., n=31). CV and BG were confirmed in fish extracts by ion trap LC-mass spectrometry (LC-MS(n)) with no discharge-atmospheric pressure chemical ionization. Average LC-MS(n) recoveries were 96.5, 96.6, and 70.2% for samples fortified with CV, LCV, and BG or LBG. The limits of detection for CV, BG, and MG were in the range of 0.07-0.24 ngg(-1) (ppb) for the two different instrumental methods. This methodology was applied to the analysis of catfish treated with CV and BG.

Optimisation of the hydrogen peroxide pre-treatment of titanium: surface characterisation and protein adsorption.

BACKGROUND: Researchers have attempted to enhance titanium osseointegration by modifying its surface properties, including via H(2)O(2) pre-treatment, with reported treatment regimes varying from minutes/hours, to weeks. OBJECTIVE: This study examined the effects of various H(2)O(2) treatments on titanium surface topography/roughness, chemical composition/oxide thickness, hydrophilicity and plasma protein adsorption. MATERIALS AND METHODS: Titanium discs were treated with 30% H(2)O(2) for 0-24 h or 1-4 weeks and subjected to atomic force microscopy (AFM), scanning electron microscopy (SEM), profilometry, X-ray photon spectroscopy and contact angle analysis. For protein adsorption, whole plasma and FITC-conjugated serum albumin were added to 0-24 h and 1-4 week H(2)O(2)-treated discs and examined by SEM and fluorescence microscopy, respectively. RESULTS: AFM, SEM and profilometry demonstrated that 1-6 h H(2)O(2)-treated discs exhibited subtle alterations in surface topography/roughness at the nanometre scale, although 24 h and 1-4 week H(2)O(2)-treated discs exhibited much greater increases in surface roughness, in the micrometre range. Maximal increases in surface oxide thickness and chemical modification were identified between 1 h-4 weeks and 3 h-4 weeks, respectively, although no increases in oxygen/titanium (O1s : Ti2p) molar ratio or in hydrophilicity were evident. Plasma and serum albumin adsorption increased on 1-24 h H(2)O(2)-treated discs, with further increases on 1-4 week H(2)O(2)-treated discs. CONCLUSIONS: Based upon the present data and previous findings, this study supports the concept that surface topography/roughness and oxide composition/thickness, are more significantly modified by H(2)O(2) treatment and more influential to protein adsorption than hydrophilicity. Additionally, it can be hypothesized that the 24 h H(2)O(2) treatment of titanium surfaces, which induced micrometre scale changes in roughness and protein adsorption, to those associated with enhanced osteoblast attachment/behaviour, mineralisation and subsequent implant osseointegration, would be most beneficial.

Neurotensin receptor levels as a function of brain aging and cognitive performance in the Morris water maze task in the rat.

The present study evaluated whether neurotensin (NT) binding sites were altered in the aged rat brain and if these alterations were related to the cognitive status of the animal. Aged (24-25 months old) Long-Evans rats were behaviorally screened using the Morris water maze task and were classified as either aged, cognitively impaired (AI) or cognitively unimpaired (AU) based on their relative performances in the task compared to young control (Y) animals. Decreases in specific [125I]NT binding were observed in the hippocampal formation, namely the dentate gyrus (DG), as well as in the septum and hypothalamus. Both aged groups also showed significant reductions in specific [125I]NT binding levels compared to the Y animals in the hippocampal CA3 sub-field, with the AI animals exhibiting the lowest levels. In the Substantia Nigra Zona Compacta (SNc) and the ventral tegmental area (VTA), specific [125I]NT binding was decreased as a function of age while binding in the paraventricular nucleus of the hypothalamus (PVNh) was decreased as a function of age and cognitive status. These alterations in the level of specific [125I] NT binding in the aged animals suggest decreases in NT receptor signaling as a function of age and potential involvement of NT-ergic systems in the etiology of age-related cognitive deficits.

The ability of fruit teas to remove the smear layer: an in vitro study of tubule patency.

OBJECTIVES: Fruit teas are know to have an erosive effect on enamel, but the effects on dentine are unknown. Lesions of dentine hypersensitivity have numerous patent dentinal tubules and the aim of this paper was to examine the ability of various fruit teas to remove the smear layer. METHODS: The erosive potential of a variety of fruit teas was assessed in the laboratory by measuring their pH and neutralisable acidity. Smeared dentine specimens were prepared from extracted teeth and the ability of each tea to remove the smear layer was assessed by measuring the diameter and area of the opened tubules and counting the number of patent tubules seen in a unit area using scanning electron microscopy. A 0.2% citric acid solution was used as a positive control. RESULTS: The pH of the fruit teas ranged from 2.98 to 3.95 and the neutralisable acidity ranged from 10.63 to 33.0 ml of 0.1 M NaOH. All the fruit teas tested were able to remove the smear layer. The mean diameter of the tubules ranged from 0.61 to 1.14 microm and the mean area ranged from 0.31 to 1.03 microm2. The number of patent tubules per specimen ranged from 13 to 121. CONCLUSION: All the fruit teas tested were found to be highly acidic and able to remove the smear layer.

Long-term effects of BIBN-99, a selective muscarinic M2 receptor antagonist, on improving spatial memory performance in aged cognitively impaired rats.

Aged Long-Evans rats were screened for spatial memory deficits using the Morris water maze task. Rats found to have impaired performance on the task (aged-impaired, AI) were then treated with a selective muscarinic M2 receptor antagonist, 5,11-dihydro-8-chloro-11-[[4-[3-[(2,2-dimethyl-1-oxopentyl)ethylamino]propyl]-1-piperidinyl]acetyl]-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one (BIBN-99; 0.5 mg/kg, s.c.), for 3 successive days while receiving additional water maze training. BIBN-99 significantly improved performance in the task during the 3 days of drug treatment. Treatment was then ceased for the remainder of the study and rats were tested again in the water maze on days 10, 17, and 24. Compared to vehicle-treated rats, enhanced performance was observed in the AI rats that had previously been treated with BIBN-99. These results indicate that BIBN-99 enhances spatial learning in AI animals and that enhanced (or long-term) memory persists in the absence of the drug. In a second experiment, a 2-month delay was imposed in between the original water maze screening and the drug treatment regime. Again, BIBN-99 significantly improved performance in AI rats. This latter study suggests that reference memory does not decay, even in an AI animal that had displayed poor learning following original water maze screening. Together, these studies help provide further insight into possible mechanism(s) of reference memory and its potential clinical usefulness.

Interlaboratory comparison of methods for the determination of incurred tilmicosin residues in bovine liver.

The objective of this study was to compare 2 methods for the determination of tilmicosin residues in bovine liver samples. Three laboratories participated in the comparison of the 2 methods. The first method was described in a New Animal Drug Application (NADA 140-929), and the second was a modification of that method in which hexane was substituted for carbon tetrachloride in one cleanup step. Each of the 3 laboratories analyzed subsamples of 10 bovine livers containing incurred tilmicosin. Residues ranged from 2.3 to 81 ppm tilmicosin in the 10 liver samples with an 11.8% relative standard deviation obtained by using both methods. In addition, fortified-control liver tissue samples were analyzed concurrently with tissues containing incurred residues by using the modified method in one of the laboratories. The fortification levels ranged from 0.3 to 112 ppm, with recoveries ranging from 76 to 92%. The results from the 3 laboratories were comparable, indicating that the modified method was not only as effective as the original NADA method, but also more desirable because of the change to a less hazardous solvent.

Effects of hippocampal lesions on patterned motor learning in the rat.

Motor skill learning in rats has been linked to cerebellar function as well as to cortical and striatal influences. The present study evaluated the contribution of the hippocampus to motor learning. Adult male rats received electrolytic lesions designed to selectively destroy the hippocampus; a sham-lesioned group of animals served as a control. The animals with hippocampal lesions acquired a patterned motor learning task as well as sham controls. In contrast, rats with hippocampal lesions were impaired in spatial, but not cued, learning in the Morris water maze. In addition, lesioned rats showed profound impairment in the novel object recognition memory task, when a 1-h delay was used between training and testing. Taken together, these results suggest that the hippocampus is not necessary during acquisition of the motor learning task.

Errors in the estimation of the distance of fall and angles of impact blood drops.

This paper presents derived equations for the estimation of the variances in the estimated distance of fall and the estimated angle of impact for blood droplets. The derived equation for the variance of the estimated distance of fall predicts that as the blood drop size approaches that of a blood droplet falling at terminal velocity, the variance of the estimate grows without limit. The derived equation for the variance in the estimated angle of impact shows that as the angle of impact approaches 90 degrees, the variance grows without limit. The validity of the equation for the estimated variance of the angle of impact was tested for five angles of impact (15, 30, 45, 60 and 75 degrees); the equation correctly predicted the observed variance up to an angle of 60 degrees.

Amyloid beta peptide levels and its effects on hippocampal acetylcholine release in aged, cognitively-impaired and -unimpaired rats.

Excessive extracellular deposition of amyloid beta (Abeta) peptide in neuritic plaques and degeneration of forebrain cholinergic neurones, which innervate the hippocampus and the neocortex, are the invariant characteristic features of Alzheimer's disease (AD). Studies of the pathological changes that characterize AD, together with several other lines of evidence, indicate that Abeta accumulation in vivo may initiate and/or contribute to the process of neurodegeneration observed in the AD brain. However, the underlying mechanisms by which Abeta peptide influences/causes degeneration of the basal forebrain cholinergic neurones in AD brains remain obscure. We reported earlier that nM concentrations of Abeta-related peptides, under acute conditions, can potently inhibit K+-evoked endogenous acetylcholine (ACh) release from the hippocampus and the cortex but not from striatum in young adult rats (J. Neurosci. 16 (1996) 1034). In the present study, to determine whether the effects of Abeta peptides alter with normal aging and/or cognitive state, we have measured Abeta1-40 levels and the effects of exogenous Abeta1-40 on hippocampal ACh release in young adult as well as aged cognitively-unimpaired (AU) and -impaired (AI) rats. Endogenous levels of Abeta(1-40) in the hippocampus are significantly increased in aged rats. Additionally, 10 nM Abeta1-40 potently inhibited endogenous ACh release from the hippocampus of the three groups of rats, but the time-course of the effects clearly indicate that the cholinergic neurones of AI rats are more sensitive to Abeta peptides than either AU or young adult rats. These results, together with earlier reports, suggest that the processing of the precursor protein of Abeta peptide alters with normal aging and the response of the cholinergic neurones to the peptide possibly varies with the cognitive status of the animals.

Limits of body mass index to detect obesity and predict body composition.

Body mass index (BMI) is commonly used to identify obesity. In this study, we determined how accurately BMI could determine body composition and identify obese from non-obese individuals. Fat-free mass and body fat were determined with bioelectrical impedance. Adiposity was calculated as body fat per body mass and as body fat divided by body height (m2). Obesity was defined as a BMI of at least 30 kg/m2 or an amount of body fat of at least 25% of total body mass for men and at least 30% for women. Obesity as defined by percentage of body fat was always present with a BMI of at least 30 kg/m2. However, 30% of men and 46% of women with a BMI below 30 kg/m2 had obesity levels of body fat. The greatest variability in the prediction of percentage of body fat and body fat divided by height (m2) from regression equations using BMI was at a BMI below 30 kg/m2. In conclusion, using impedance-derived body-fat mass as the criterion, people with BMI of at least 30 kg/m2 are obese. However, significant numbers of people with a BMI below 30 kg/m2 are also obese and thus misclassified by BMI. Percent of body fat and body fat divided by height (m2) are predictable from BMI, but the accuracy of the prediction is lowest when the BMI is below 30 kg/m2. Therefore, measurement of body fat is a more appropriate way to assess obesity in people with a BMI below 30 kg/m2.

The current status of microscopical hair comparisons.

Although the microscopical comparison of human hairs has been accepted in courts of law for over a century, recent advances in DNA technology have called this type of forensic examination into question. In a number of cases, post-conviction DNA testing has exonerated defendants who were convicted in part on the results of microscopical hair comparisons. A federal judge has held a Daubert hearing on the microscopical comparison of human hairs and has concluded that this type of examination does not meet the criteria for admission of scientific evidence in federal courts. A review of the available scientific literature on microscopical hair comparisons (including studies conducted by the Royal Canadian Mounted Police and the Federal Bureau of Investigation) leads to three conclusions: (1) microscopical comparisons of human hairs can yield scientifically defensible conclusions that can contribute to criminal investigations and criminal prosecutions, (2) the reliability of microscopical hair comparisons is strongly affected by the training of the forensic hair examiner, (3) forensic hair examiners cannot offer estimates of the probability of a match of a questioned hair with a hair from a randomly selected person. In order for microscopical hair examinations to survive challenges under the U.S. Supreme Court's Daubert decision, hair microscopists must be better trained and undergo frequent proficiency testing. More research on the error rates of microscopical hair comparisons should be undertaken, and guidelines for the permissible interpretations of such comparisons should be established. Until these issues have been addressed and satisfactorily resolved, microscopical hair comparisons should be regarded by law enforcement agencies and courts of law as merely presumptive in nature, and all microscopical hair comparisons should be confirmed by nuclear DNA profiling or mitochondrial DNA sequencing.

Founding editorial--forensics and TheScientificWorld.

At the beginning of a new millennium it seems a good idea to stop for a moment and take stock of the current state of forensic science. As a field of scientific research and scientific application, forensic science is a little more than a century old. Forensic science may be said to have begun in 1887 with the simultaneous publication of A. Conan Doyle's A Study in Scarlet and Hans Gross's Handbuch f1/4r Untersuchungsrichter. Conan Doyle's novel introduced to the world the character of Sherlock Holmes, whose literary career would popularize the use of physical evidence in criminal investigations. Gross's manual for examining magistrates suggests ways in which the expertise of chemists, biologists, geologists, and other natural scientists could contribute to investigations. Gross's book was translated into a number of languages and went through various updated editions during the course of the century. The intervening century saw the development and application of fingerprinting, firearm and tool mark identification, forensic chemistry, forensic biology, forensic toxicology, forensic odontology, forensic pathology, and forensic engineering. Increasingly, the judicial systems of the industrial nations of the world have come to rely upon the expertise of scientists in a variety of disciplines. In most advanced countries, virtually all criminal prosecutions now involve the presentation of scientific testimony. This has had the beneficial effect of diminishing the reliance of courts on eyewitness testimony and defendant confessions.

Increase in AMPA receptors in aged memory-impaired rats is not associated with increase in monoamine oxidase B levels.

Aged rats may be behaviorally classified as either cognitively impaired or unimpaired based upon their performance in the Morris water maze task. In aged Long-Evans rats, emergence of functional deficits has been related to the increase in the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subtype in most hippocampal subfields, not observed in other brain structures. As AMPA receptors expressed in astrocytes may participate in the delayed and long-term glial response to injury, we investigated whether astrocytes participate in the increase of AMPA receptor observed in these aged rats. To this end, distribution of monoamine oxidase B, used as an astroglial marker, was characterized by quantitative autoradiography in the hippocampus and septum of young adults (six months) and aged (24-25 months) rats using [3H]lazabemide. Specific binding to brain sections of young, aged unimpaired, and aged impaired animals were calculated densitometrically. Compared to young animals, all hippocampal subfields in the aged unimpaired group showed a significant age-related increased labeling, which was not present in the aged impaired group. This contrasts with the increased glial transcription described in this last group. We propose that increase in AMPA receptors in the aged memory-impaired animals may be related to an atypic astrocytic reactivity.

Comparing the additive composition of smokeless gunpowder and its handgun-fired residues.

Detecting the use of handguns via the determination of the organic additives in smokeless gunpowder residues (OGSR) presents a promising alternative to primer metal residue analysis. Compositional analysis of the gunpowder additives nitroglycerin, diphenylamine, and ethyl centralite provides information that can associate residue samples with unfired gunpowder. We evaluated the composition of seven reloading smokeless gunpowders, both in bulk and as single particles, by ultrasonic solvent extraction/capillary electrophoresis. Handgun-fired residues obtained from three common weapon calibers loaded with the known reloading powders were compared with the unfired powders. In general, the composition of the residues was similar to that found in the unfired powders. For double-base powders, comparing the ratio of the propellant (P) to the total amount of stabilizer (S) for both residue and gunpowder samples proved to be a useful measurement for identification. This P/S ratio demonstrated that the additives in the residues did not greatly change relative to the unfired powder, providing a useful indicator to aid in forensic powder and residue evaluation.