Mikronuklei als Indikatoren nach Bestrahlung und Charakterisierung ihrer Entstehung mit Hilfe von DNA-Sonden

Ein einfacher Test, um chromosomale Schäden zu messen, ist die Bestimmung von Mikronuklei. Es sind vom Zellkern abstammende chromosomale Strukturen, die nach einer Zellteilung nicht in die Tochterzellkerne aufgenommen wurden und im Cytoplasma als Chromatinpartikel erscheinen. Voraussetzung für die Mikronukleusexpression ist die Kernteilung. Um zu überprüfen, ob eine Zelle die Mitose durchlaufen hat, setzt man Cytochalasin B ein, das in bestimmten Konzentrationsbereichen die Eigenschaft hat, die Zellteilung zu unterbinden, eine Kernteilung jedoch zuzulassen. Dadurch sind die Zellen, die eine Mitose durchlaufen haben, daran zu erkennen, daß sie zwei Zellkerne aufweisen. Mikronuklei werden ausschließlich in solchen Zellen gezählt. Es ist bekannt, daß die Frequenz strahleninduzierter Mikronuklei strahlenart- und dosisabhängig ist. Untersuchungen der Mikronukleusraten in menschlichen Lymphocyten zeigten, daß nach in vitro und in vivo Bestrahlung keine unterschiedlichen Strahleneffekte zu beobachten sind. Frühere Untersuchungen haben ergeben, daß spontan entstehende Mikronuklei häufig aus ganzen Chromosomen bestehen. Dagegen sind die strahleninduzierten Mikronuklei vorwiegend auf azentrische Fragmente zurückzuführen. Durch Erweiterung des Mikronukleustestes mit der Fluoreszenz-in-situ-Hybridisierungs-Technik (FISH) kann man die Empfindlichkeit des Testes steigern. Mit Hilfe von DNA-Sonden gegen Centromere können die Mikronuklei dahingehend charakterisiert werden, ob sie ganze Chromosomen oder nur azentrische Fragmente enthalten. Im Rahmen dieser Untersuchungen wurde die Nützlichkeit des Mikronukleustestes in der biologischen Dosimetrie überprüft. Als Schwerpunkt wurde die Möglichkeit der Verwendung des Testes im niedrigen Dosisbereich (unter 0,5 Gy) festgelegt. Zu diesem Zweck wurde das Blut von Kontrollspendern und auch von ehemaligen Uranbergarbeitern der Wismut AG untersucht. Bei den Uranbergarbeitern handelte sich um Arbeiter, die in den Jahren 1946-1990 durch ungenügenden Strahlenschutz im Uranbergbau sehr hohen Strahlenexpositionen ausgesetzt waren. Die Wismut-Arbeiter wurden in zwei unterschiedliche Gruppen unterteilt. Zu der ersten Gruppe gehörten die Arbeiter, die nach der Tätigkeit bei der Wismut AG bis jetzt kein Karzinom entwickelt haben. Die zweite Gruppe repräsentiert die Wismut-Arbeiter, bei denen ein Bronchialkarzinom festgestellt wurde. In jeder untersuchten Gruppe (auch bei Kontrollspendern) wurden beide Varianten des Mikronukleustestes (Mikronukleustest mit Cytochalasin B-Technik und Centromer-Mikronukleustest) durchgeführt. Zur Ermittlung der Dosiswirkungsbeziehung wurde das Blut von Kontrollspendern in vitro mit verschiedenen Dosen Röntgenstrahlen exponiert. Eine Analyse von Chromosomenaberrationen diente dazu, weitere chromosomale Strahlenschäden zu untersuchen. Ergebnisse: I. Mikronukleus-Cytochalasin B-Test 1. Es wurden keine signifikanten Unterschiede in den spontanen Mikronukleushäufigkeiten zwischen gesunden Spendern, Wismut-Spendern ohne Tumor und Wismut-Spendern mit Tumor beobachtet. 2. In bestrahlten Lymphocyten der gesunden Spender mit Dosen unter 0,5 Gy wurden signifikante Unterschiede gegenüber der Spontanhäufigkeit nur auf dem Populationsniveau ermittelt. 3. In bestrahlten Lymphocyten der gesunden Spender mit Dosen ab 0,5 Gy wurden signifikante Unterschiede bei den Mikronukleushäufigkeiten gegenüber der Spontanhäufigkeit auf dem individuellen Niveau festgestellt. II. Centromer-Mikronukleustest 1. Spontane Mikronuklei sind vorwiegend auf ganze Chromosomen zurückzuführen. 2. Bei beruflich exponierten Personen (Wismut-Arbeiter) war der Anteil an Centromer-negativen Mikronuklei signifikant erhöht gegenüber nicht exponierten Spendern. 3. Die relative Anzahl der Centromer-positiven Mikronuklei in unbestrahlten Lymphocyten war zwischen allen untersuchten Gruppen unterschiedlich. 4. Nach in vitro Strahlenexpositionen wurde eine dosisabhängige Abnahme der Centromer-positiven Mikronuklei bei gesunden Spendern (schon ab 0,1 Gy) beobachtet. 5. Mit dem Centromer-Mikronukleustest wurden frühere berufliche Strahlenexpositionen qualitativ erfaßt. III. Statistische Kombination der Ergebnisse des Mikronukleus-Cytochalasin B-Testes und Centromer-Mikronukleustestes führt zu der Steigerung der Empfindlichkeit des Mikronukleustestes auf dem individuellen Niveau. IV. Chromosomenaberrationen Auswertung 1. Die Aberrationshäufigkeit in Lymphocyten der Wismut-Arbeiter mit Tumor war signifikant höher als in Lymphocyten gesunder Spender. 2. Es wurden keine Korrelationen zwischen Mikronukleushäufigkeiten und Chromosomenaberrationen bei gesunden Spendern und Wismut-Arbeitern mit Tumor ermittelt. 3. Es wurde eine deutliche negative Korrelation zwischen Centromer-positiven Mikronuklei und Chromosomenaberrationen beobachtet

An Objective Assessment of the Variability in Number of Drops per Bottle of Glaucoma Medication

Background: The purpose of this study is to evaluate the number of eyedrops available per bottle of a variety of commonly prescribed glaucoma medications. Methods: Six bottles of each glaucoma medication were tested: three each in the vertical and horizontal orientations. Bottles were housed in a customized force gauge apparatus designed to mimic ballpoint fingertip contact with a bottle. At a standard rate, all drops were expressed from each bottle and counted with an automated drop counter. Simultaneously, bottle volume was measured and drop size and number were also estimated. The main outcome measures were: total number of drops, volume per bottle and drops per milliliter (mL) of glaucoma medication. Results: A total of 192 bottles from 32 bottle designs and manufacturers were tested. Twenty-two of the 32 bottle designs had a significantly different mean number of drops in the vertical and horizontal positions, with 10 designs have more drops dispensed in the horizontal orientation and 12 in the vertical orientation. Six of the 32 bottle designs had a significantly different mean total bottle volume in the vertical and horizontal positions, with all designs having greater volume in the vertical position. An adjusted ratio of mean number of drops/mean bottle volume demonstrated a range from 20.9 drops/mL to 40.8 drops/mL. Conclusions: There is significant variability in drops and volume available per bottle of glaucoma medication depending on both the bottle position and manufacturer. These data point to the need for circumspection in prescribing glaucoma medications and caution in evaluating therapeutic outcomes

A Case Study: Teachers’ Perceptions Of The Influence Of Proessional Development On Self-Efficacy Related To Implementing A Balanced Literacy Model

Researchers have known for several years that when a child is reading below proficiency by the end of third grade, it is likely the child will continue to struggle learning to read through the child’s academic career (Dickinson & Porche, 2011; Wasik & Hindman, 2011). Yet, the problem of children falling below third grade reading proficiency continues. If teaching a child to read is a moral purpose, and it is essential for a child to read proficiently by the end of third grade, then literacy instruction in kindergarten through third grade is pivotal. In this case study, the researcher sought to illustrate the beliefs and literacy teaching practices of teacher participants within one public school district that provided training in Balanced Literacy components and required teachers to implement Balanced Literacy. Specific research questions explored teacher perceptions of self-efficacy and preparedness to implement Balanced Literacy. A purposive convenience sample was used to identify Kindergarten, Grade 1, and Grade 2 teachers within the school district who taught literacy to their students in the 2016-2017, 2017-2018 and 2018-2019 school years. Individual interviews and classroom observations were transcribed and coded by discrete idea relative the study’s conceptual and theoretical frameworks: Fountas and Pinnell’s (1996) Balanced Literacy framework and Bandura’s (1997) sources of self-efficacy theories. Findings indicated teacher self-efficacy varied across Balanced Literacy components. Teachers felt most prepared to teach Word Study. Teachers benefited from modeling by the Reading Specialist, felt encouraged by feedback, and desired support in differentiating instruction. Word Study instruction had high alignment to the Balanced Literacy Model provided by the school district, and Guided Reading and Writing Workshop had low alignment. These findings validate the support the school district provides for teachers and directs school leaders in developing professional development needs to support stronger reading teachers

The TNFα-Transgenic Rat: Hippocampal Synaptic Integrity, Cognition, Function, and Post-Ischemic Cell Loss

The cytokine, tumor necrosis factor α (TNFα), is a key regulator of neuroinflammation linked to numerous neurodegenerative conditions and diseases. The present study used transgenic rats that overexpress a murine TNFα gene, under the control of its own promoter, to investigate the impact of chronically elevated TNFα on hippocampal synaptic function. Neuronal viability and cognitive recovery in TNFα Tg rats were also determined following an ischemic insult arising from reversible middle cerebral artery occlusion (MCAO). Basal CA3-CA1 synaptic strength, recorded in acute brain slices, was not significantly different between eight-week-old TNFα Tg rats and non-Tg rats. In contrast, slices from TNFα Tg rats showed significantly greater levels of long-term potentiation (LTP) in response to 100 Hz stimulation, suggesting that synaptic networks may be hyperexcitable in the context of elevated TNFα. Cognitive and motor deficits (assessed on the Morris Water Maze and Rotarod task, respectively) were present in TNFα Tg rats in the absence of significant differences in the loss of cortical and hippocampal neurons. TNF overexpression exacerbated MCAO-dependent deficits on the rotarod, but ameliorated cortical neuron loss in response to MCAO

Evaluation of Unmanned Radar Installations

Several unmanned radar devices were installed on I75 in northern Kentucky in an attempt to reduce speeds. It was assumed that drivers use radar detectors to exceed the speed limit with a resulting variance between their speeds and others in the traffic stream. Therefore, a reduction in overall speeds and variance was expected to reduce the probability of accidents. Historical data indicated an unusually high accident rate for the study area. Emphasis was placed on collection and analysis of speed-related data. In addition, a survey of radar detector usage was made and accident patterns were documented. Speed measures analyzed included mean speed, standard deviation in speed, numbers of vehicles exceeding specified speed levels, and 85th percentile speed. Results indicate that unmanned radar was an effective means of reducing the number of vehicles traveling at excessive speeds. The differences in mean speeds were small and the impact of unmanned radar was less obvious than it was for the percentage of vehicles exceeding speed levels of 65, 70, 75, and 80 mph. The speeds of vehicles with radar detectors decreased significantly as a result of unmanned radar while the speeds of vehicles without detectors were not affected. Radar detector usage was found to be 42 percent in trucks and 11 percent in cars. When comparing accident data three years before and one year after truck diversion and unmanned radar installations, there was a reduction in truck-related and speed-related accidents

Stochastic models of kleptoparasitism

In this paper, we consider a model of kleptoparasitism amongst a small group of individuals, where the state of the population is described by the distribution of its individuals over three specific types of behaviour (handling, searching for or fighting over, food). The model used is based upon earlier work which considered an equivalent deterministic model relating to large, effectively infinite, populations. We find explicit equations for the probability of the population being in each state. For any reasonably sized population, the number of possible states, and hence the number of equations, is large. These equations are used to find a set of equations for the means, variances, covariances and higher moments for the number of individuals performing each type of behaviour. Given the fixed population size, there are five moments of order one or two (two means, two variances and a covariance). A normal approximation is used to find a set of equations for these five principal moments. The results of our model are then analysed numerically, with the exact solutions, the normal approximation and the deterministic infinite population model compared. It is found that the original deterministic models approximate the stochastic model well in most situations, but that the normal approximations are better, proving to be good approximations to the exact distribution, which can greatly reduce computing time

Lifelong Bilingualism Maintains Neural Efficiency for Cognitive Control in Aging

Recent behavioral data have shown that lifelong bilingualism can maintain youthful cognitive control abilities in aging. Here, we provide the first direct evidence of a neural basis for the bilingual cognitive control boost in aging. Two experiments were conducted, using a perceptual task-switching paradigm, including a total of 110 participants. In Experiment 1, older adult bilinguals showed better perceptual switching performance than their monolingual peers. In Experiment 2, younger and older adult monolinguals and bilinguals completed the same perceptual task-switching experiment while functional magnetic resonance imaging (fMRI) was performed. Typical age-related performance reductions and fMRI activation increases were observed. However, like younger adults, bilingual older adults outperformed their monolingual peers while displaying decreased activation in left lateral frontal cortex and cingulate cortex. Critically, this attenuation of age-related over-recruitment associated with bilingualism was directly correlated with better task-switching performance. In addition, the lower blood oxygenation level-dependent response in frontal regions accounted for 82% of the variance in the bilingual task-switching reaction time advantage. These results suggest that lifelong bilingualism offsets age-related declines in the neural efficiency for cognitive control processes

Spectroscopic and quartz crystal microbalance (QCM) characterisation of protein-based MIPs

We have studied acrylamide-based polymers of varying hydrophobicity (acrylamide, AA; N-hydroxymethylacrylamide, NHMA; N-isopropylacrylamide, NiPAm) for their capability of imprinting protein. Rebinding capacities (Q) from spectroscopic studies were highest for bovine haemoglobin (BHb) MIPs based on AA, Q = 4.8 ± 0.21 76 ± 0.5%). When applied to the QCM sensor as thin-film MIPs, NHMA MIPs were found to exhibit best discrimination between MIP and non-imprinted control polymer (NIP) in the order of NiPAm < AA < NHMA. The extent of template removal and rebinding, using both crystal impedance and frequency measurements, demonstrated that 10% (w/v):10% (v/v) sodium dodecyl sulphate:acetic acid (pH 2.8) was efficient at eluting template BHb (with 80 ± 10% removal). Selectivity studies of NHMA BHb-MIPs revealed higher adsorption and selective recognition properties to BHb (64.5 kDa) when compared to non-cognate BSA (66 kDa), myoglobin (Mb, 17.5 kDa), lysozyme (Lyz, 14.7 kDa) thaumatin (Thau, 22 kDa) and trypsin (Tryp, 22.3 kDa). The QCM gave frequency shifts of ∼1500 ± 50 Hz for template BHb rebinding in both AA and NHMA MIPs, whereas AA-based MIPs exhibited an interference signal of ∼2200 ± 50 Hz for non-cognate BSA in comparison to a ∼500 ± 50 Hz shift with NHMA MIPs. Our results show that NHMA-based hydrogel MIP are superior to AA and NIPAM

Determination of protein binding affinities within hydrogel-based molecularly imprinted polymers (HydroMIPs)

Hydrogel-based molecularly imprinted polymers (HydroMIPs) were prepared for several proteins (haemoglobin, myoglobin and catalase) using a family of acrylamide-based monomers. Protein affinity towards the HydroMIPs was investigated under equilibrium conditions and over a range of concentrations using specific binding with Hill slope saturation profiles. We report HydroMIP binding affinities, in terms of equilibrium dissociation constants (Kd) within the micro-molar range (25 ± 4 mM, 44 ± 3 mM, 17 ± 2 mM for haemoglobin, myoglobin and catalase respectively within a polyacrylamide-based MIP). The extent of non-specific binding or cross-selectivity for non-target proteins has also been assessed. It is concluded that both selectivity and affinity for both cognate and non-cognate proteins towards the MIPs were dependent on the concentration and the complementarity of their structures and size. This is tentatively attributed to the formation of protein complexes during both the polymerisation and rebinding stages at high protein concentrations. We have used atomic force spectroscopy to characterize molecular interactions in the MIP cavities using protein-modified AFM tips. Attractive and repulsive force curves were obtained for the MIP and NIP (non-imprinted polymer) surfaces (under protein loaded or unloaded states). Our force data suggest that we have produced selective cavities for the template protein in the MIPs and we have been able to quantify the extent of non-specific protein binding on, for example, a non-imprinted polymer (NIP) control surface