The Application of CRISPR Technology to High Content Screening in Primary Neurons

Axon growth is coordinated by multiple interacting proteins that remain incompletely characterized. High content screening (HCS), in which manipulation of candidate genes is combined with rapid image analysis of phenotypic effects, has emerged as a powerful technique to identify key regulators of axon outgrowth. Here we explore the utility of a genome editingapproach referred to as CRISPR (Clustered Regularly Interspersed Palindromic Repeats) for knockout screening in primary neurons. In the CRISPR approach a DNA-cleaving Cas enzyme is guided to genomic target sequences by user-created guide RNA (sgRNA), where it initiates a double-stranded break that ultimately results in frameshift mutation and loss of protein production. Using electroporation of plasmid DNA that co-expresses Cas9enzyme and sgRNA, we first verified the ability of CRISPR targeting to achieve protein-level knockdown in cultured postnatal cortical neurons. Targeted proteins included NeuN (RbFox3) and PTEN, a well-studied regulator of axon growth. Effective knockdown lagged at least four days behind transfection, but targeted proteins were eventually undetectable by immunohistochemistry in \u3e 80% of transfected cells. Consistent with this, anti-PTEN sgRNA produced no changes in neurite outgrowth when assessed three days post-transfection. When week-long cultures were replated, however, PTEN knockdown consistently increased neurite lengths. These CRISPR-mediated PTEN effects were achieved using multi-well transfection and automated phenotypic analysis, indicating the suitability of PTEN as a positive control for future CRISPR-based screening efforts. Combined, these data establish an example of CRISPR-mediated protein knockdown in primary cortical neurons and its compatibility with HCS workflows

Lung analysis in children mechanically ventilated with atelectasis after cardiac surgery

El objetivo de este estudio es asociar la área sana del pulmón (evaluado mediante radiografía) a los datos de mecánica respiratoria en niños en el postoperatorio de cirugía cardiaca, con atelectasia en ventilación mecánica en el modo asistido controlado. En el total, fueron seleccionados 46 niños, de los cuales 16 fueron excluidos debido a las ondas respiratorias irregulares o a la falta de datos de la gasometria arterial. Se analizó un grupo de 30 niños en el modo asistido controlado, de los cuales 10 han desarrollado atelectasia. Los datos fueron analizados antes y después del inicio de la atelectasia, y la mecánica respiratoria se correlacionó a las medidas del área del pulmón. En los 30 niños inicialmente sin complicación pulmonar fueron analizados los datos de la gasometria arterial para comprobar posibles cambios debido a los ciclos asistidos. La atelectasia puede modificar algunos parámetros de la mecánica respiratoria. En la asociación del área saludable del pulmón con la mecánica respiratoria, los resultados de correlación de Spearman demostraron significancia estadística entre el área del pulmón con la resistencia de las vías aéreas (ρ=-0,648 y p=0,043). Los resultados mostraron que es posible el análisis de las ondas de mecánica respiratoria a través de la selección de los ciclos controlados, puesto que no hubo alteración significativa en el potencial de hidrógeno iónico. El análisis de la mecánica respiratoria permite comprobar la función pulmonar y las posibles lesiones pulmonares. Es posible utilizar el análisis de la mecánica respiratoria diariamente en estos niños, permitiendo obtener informaciones importantes de la función pulmonar. El estudio también mostró que en el modo de ventilación controlada es posible evaluar la mecánica respiratoria.The main objective of this study is to associate the healthy area of the lung (evaluated by radiography) with the data of respiratory mechanics in children with atelectasis after cardiac surgery, under mechanical ventilation in the assisted controlled mode. Altogether, 46 children were selected, but 16 were excluded due to irregular respiratory waves or lack of the data on arterial blood gases. A group of 30 children under assisted controlled mode were analyzed, and 10 from this group developed atelectasis. The data were analyzed before and after the onset of atelectasis, and respiratory mechanics was correlated to radiography. We also analyzed the data related to arterial blood gas of these children - who initially had no pulmonary complication - to verify possible changes due to assisted controlled cycles. Atelectasis may modify some parameters of respiratory mechanics. In the association of the healthy area of the lung with the respiratory mechanics, the Spearman correlation results showed statistical significance of the lung area with airway resistance (ρ=−0.648 and p=0.043). Our results show that it is possible to analyze respiratory mechanics waves by selecting controlled cycles in the assisted controlled mode, since we found insignificant changes in potential ionic hydrogen. The analysis of respiratory mechanics allows checking lung function and undesired lung injuries; the analysis of respiratory mechanics can be daily performed in these children to have important information on the pulmonary function. Our research also showed that under the assisted controlled mode is also possible to evaluate respiratory mechanics.O objetivo deste estudo é associar a área saudável do pulmão (avaliada pela radiografia) aos dados de mecânica respiratória em crianças no pós-operatório de cirurgia cardíaca, com atelectasia em ventilação mecânica no modo assistido controlado. No total, foram selecionadas 46 crianças, das quais 16 foram excluídas devido às ondas respiratórias irregulares ou à falta de dados da gasometria arterial. Foi analisado um grupo de 30 crianças em modo assistido controlado, sendo que 10 crianças desenvolveram atelectasia. Os dados foram analisados antes e após o início da atelectasia, e a mecânica respiratória foi correlacionada às medidas da área do pulmão. Nas 30 crianças, inicialmente sem complicação pulmonar, foram analisados os dados da gasometria arterial para verificar possíveis mudanças devido aos ciclos assistidos. A atelectasia pode modificar alguns parâmetros da mecânica respiratória. Na associação da área saudável do pulmão com a mecânica respiratória, os resultados da correlação de Spearman mostraram significância estatística entre a área do pulmão com a resistência das vias aéreas (ρ=-0,648 e p=0,043). Os resultados demonstraram que é possível a análise das ondas de mecânica respiratória através da seleção dos ciclos controlados, uma vez que não houve alteração significativa no potencial de hidrogênio iônico. A análise da mecânica respiratória permite verificar a função pulmonar e as possíveis lesões pulmonares. A análise da mecânica respiratória pode ser usada diariamente nessas crianças, permitindo obter informações importantes da função pulmonar. O estudo também mostrou que no modo ventilatório assistido controlado também é possível avaliar a mecânica respiratória

Improved single-chain transactivators of the Tet-On gene expression system

BACKGROUND: The Tet-Off (tTA) and Tet-On (rtTA) regulatory systems are widely applied to control gene expression in eukaryotes. Both systems are based on the Tet repressor (TetR) from transposon Tn10, a dimeric DNA-binding protein that binds to specific operator sequences (tetO). To allow the independent regulation of multiple genes, novel Tet systems are being developed that respond to different effectors and bind to different tetO sites. To prevent heterodimerization when multiple Tet systems are expressed in the same cell, single-chain variants of the transactivators have been constructed. Unfortunately, the activity of the single-chain rtTA (sc-rtTA) is reduced when compared with the regular rtTA, which might limit its application. RESULTS: We recently identified amino acid substitutions in rtTA that greatly improved the transcriptional activity and doxycycline-sensitivity of the protein. To test whether we can similarly improve other TetR-based gene regulation systems, we introduced these mutations into tTA and sc-rtTA. Whereas none of the tested mutations improved tTA activity, they did significantly enhance sc-rtTA activity. We thus generated a novel sc-rtTA variant that is almost as active and dox-sensitive as the regular dimeric rtTA. This variant was also less sensitive to interference by co-expressed TetR-based tTS repressor protein and may therefore be more suitable for applications where multiple TetR-based regulatory systems are used. CONCLUSION: We developed an improved sc-rtTA variant that may replace regular rtTA in applications where multiple TetR-based regulatory systems are used

Perbedaan Asupan Lemak, Lingkar Pinggang dan Persentase Lemak Tubuh pada Wanita Dislipidemia dan Non Dislipidemia

Differences of fat intake, waist circumference and percentage of bodt fat in dyslipidemia and non dyslipidemia adult women: Heart disease is the leading cause of death in several countries in the world . One of the major risk factors for heart disease is dyslipidemia . Dyslipidemia is a disorder of lipid metabolism characterized by an increase or decrease in plasma lipid fractions . Dyslipidemia has a strong relationship with the occurrence of central obesity . The purpose of this study was to analyze differences in the intake of fat , waist circumference and body fat percentage in dyslipidemia and non dyslipidemia adult women. This research is analytic study with cross sectional approach . The population in this study were adult women who examined their lipid profile in December 2013 in the Clinical Laboratory Cito Indraprasta Semarang . The total sample was 32 people . Independent test analysis of the differences using t-test for variables waist circumference and Mann Whitney test for variable fat intake and body fat percentage to 95 % and a significance level of 5% error. The results showed 17 adult women ( 53.1 % ) and 15 female adult dyslipidemia ( 46.9 % ) non- dyslipidemia. Average intake of fat, waist circumference and percentage body fat in adult women dyslipidemia higher than non dyslipidemia in adult women. Analysis of statistical tests showed difference in fat intake , waist circumference and body fat percentage in women adult dyslipidemia and non dyslipidemia (p value, respectively p = 0.002, p = 0.0001 and p = 0.0001

SmartEx: a case study on user profiling and adaptation in exhibition booths

An investigation into user profiling and adaptation with exhibition booth as a case study is reported. First a review of the field of exhibitions and trade fairs and a summary introduction to adaptation and profiling are given. We then introduce three criteria for the evaluation of exhibition booth: effectiveness, efficiency and affect. Effectiveness is related the amount of information collected, efficiency is a measurement of the time taken to collect the information, and affect is the perception of the experience and the mood booth visitors have during and after their visit. We have selected these criteria to assess adaptive and profiled exhibition booths, we call smart exhibition (SmartEx). The assessment is performed with an experiment with three test conditions (non-profiled/non adaptive, profiled/non-adaptive and profiled adaptive presentations). Results of the experiment are presented along discussion. While there is significant improvements of effectiveness and efficiency between the two-first test conditions, the improvement is not significant for the last test condition, for reasons explained. As for the affect, the results show that it has an under-estimated importance in people minds and that it should be addressed more carefully

Loneliness and the Emotional Experience of Absence

In this paper, we develop an analysis of the structure and content of loneliness. We argue that this is an emotion of absence-an affective state in which certain social goods are regarded as out of reach for the subject of experience. By surveying the range of social goods that appear to be missing from the lonely person's perspective, we see what it is that can make this emotional condition so subjectively awful for those who undergo it, including the profound sense of being unable to realise oneself, in collaboration with others

A test of the household income process using consumption and wealth data

The evolution of household income can be explained almost equally well by rival models. However, rival models have very different implications for other household behaviours, such as consumption. I therefore test between two prominent models in the UK using panel data on consumption and wealth, as well as income, over 1991–2006. To operate the test, I show that long-lived income shocks transmit far less than one-for-one through to consumption, and particularly so for younger households. I then compare these estimates of transmission with estimates of households’ ability to smooth shocks, captured by the data on wealth. Conditional on the suitability of the consumption model, my estimates provide evidence against the restricted income process (RIP) and in favour of an alternative heterogeneous income process (HIP). This finding also explains why cross-sectional consumption inequality grew slowly over the period even though the variance of long-lived shocks was high. Finally, I conclude that it is important to consider mean reversion of shocks when constructing life-cycle consumption models

The Vehicle, Fall 1992

Table of Contents DeconstructivismPeter F. Essigpage 5 Homecoming Pep RallyPeter F. Essigpage 6 McAfee GymnasiumWalt Howardpage 7 Morton ParkAnn Moutraypage 9 Why The Willows WeepPeter F. Essigpage 10 UntitledStephen P. Carmodypage 10 A Stranger\u27s MorningBen Hausmannpage 11 deMONSTERative pronounsJoAnna Wolaverpage 12 2.5%Jill S. Pilonpage 13 The BottleStacey Kruegerpage 14 Suppression Jean K. Graypage 15 ProgressStacey Kruegerpage 16 Daily LessonsJennifer Moropage 17 Sunset TheaterMichelle R. Hokepage 20 Eagle GT\u27sJarrod T. Shieldspage 21 New HouseRandy Lisspage 22 UntitledStephen P. Carmodypage 23 Renting Classics on a Saturday NightNancy Jamespage 24 UntitledJacqueline Hallpage 25 Alone While He SleepsSandy Beauchamppage 26 Sand and SeaThomas Schnarrepage 27 loveMichelle R. Hokepage 28 Backward Ass Junkie FunkSandy Beauchamppage 28 These Things You KeepTom McGrathpage 29 Springhill CrestRobert M. Reutherpage 30 The Pass OverLarry Irvinpage 31 The Stolen ChildTom McGrathpage 32 Before the Recycling KickWalt Howardpage 37 Authors\u27 Pagepage 38https://thekeep.eiu.edu/vehicle/1058/thumbnail.jp

Developing a new teaching approach for the chemical bonding concept aligned with current scientific and pedagogical knowledge

The traditional pedagogical approach for teaching chemical bonding is often overly simplistic and not aligned with the most up-to-date scientific models. As a result, high-school students around the world lack fundamental understanding of chemical bonding. In order to improve students' understanding of this concept, it was essential to propose a systemic treatment, namely, revising the scientific content, the pedagogical approach, and the assessment methods regarding this concept. Therefore, the main goal of this study was to build a conceptual framework that provides an advanced scientific and pedagogical foundation regarding the chemical bonding concept—one that will guide chemistry curriculum developers as well. A conceptual framework for a new teaching approach was constructed with lead-chemistry teachers, science (chemistry) educators, and research chemists. We suggest that chemical bonding should be taught based on elemental principles and by using the idea of a continuum of bond strengths. Our process includes the formulation of learning goals aligned with current scientific knowledge. Moreover, we suggest that constructing assessment tasks on carefully specified learning goals, which are described in terms of learning performances , may enable educators to foster and examine much deeper levels of students' understanding. © 2007 Wiley Periodicals, Inc. Sci Ed 91: 579–603, 2007Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56057/1/20201_ftp.pd