Ligation dependent allele specific quantification (LASQ) of CFTR cDNA on the LightCycler using MLPA hybridization probes

BACKGROUND: As for Cystic Fibrosis (CF) and many other hereditary diseases there is still a lack in understanding the relationship between genetic (e.g. allelic) and phenotypic diversity. Therefore methods which allow fine quantification of allelic proportions of mRNA transcripts are of high importance. METHODS: We used either genomic DNA (gDNA) or total RNA extracted from nasal cells as starting nucleic acid template for our assay. The subjects included in this study were 9 CF patients compound heterozygous for the F508del mutation and each one F508del homozygous and one wild type homozygous respectively. We established a novel ligation based quantification method which allows fine quantification of the allelic proportions of ss and ds CFTR cDNA. To verify reliability and accuracy of this novel assay we compared it with semiquantitative fluorescent PCR (SQF-PCR). RESULTS: We established a novel assay for allele specific quantification of gene expression which combines the benefits of the specificity of the ligation reaction and the accuracy of quantitative real-time PCR. The comparison with SQF-PCR clearly demonstrates that LASQ allows fine quantification of allelic proportions. CONCLUSION: This assay represents an alternative to other fine quantitative methods such as ARMS PCR and Pyrosequencing.

DESIGN AND IMPLEMENT DYNAMIC PROGRAMMING BASED DISCRETE POWER LEVEL SMART HOME SCHEDULING USING FPGA

With the development and capabilities of the Smart Home system, people today are entering an era in which household appliances are no longer just controlled by people, but also operated by a Smart System. This results in a more efficient, convenient, comfortable, and environmentally friendly living environment. A critical part of the Smart Home system is Home Automation, which means that there is a Micro-Controller Unit (MCU) to control all the household appliances and schedule their operating times. This reduces electricity bills by shifting amounts of power consumption from the on-peak hour consumption to the off-peak hour consumption, in terms of different “hour price”. In this paper, we propose an algorithm for scheduling multi-user power consumption and implement it on an FPGA board, using it as the MCU. This algorithm for discrete power level tasks scheduling is based on dynamic programming, which could find a scheduling solution close to the optimal one. We chose FPGA as our system’s controller because FPGA has low complexity, parallel processing capability, a large amount of I/O interface for further development and is programmable on both software and hardware. In conclusion, it costs little time running on FPGA board and the solution obtained is good enough for the consumers.

DESIGN AND IMPLEMENTATION OF HOME USE PORTABLE SMART ELECTRONICS

The widespread of low cost embedded electronics makes it easier to implement the smart devices that can understand either the environment or the user behaviors. The main object of this project is to design and implement home use portable smart electronics, including the portable monitoring device for home and office security and the portable 3D mouse for convenient use. Both devices in this project use the MPU6050 which contains a 3 axis accelerometer and a 3 axis gyroscope to sense the inertial motion of the door or the human hands movement. For the portable monitoring device for home and office security, MPU6050 is used to sense the door (either home front door or cabinet door) movement through the gyroscope, and Raspberry Pi is then used to process the data it receives from MPU6050, if the data value exceeds the preset threshold, Raspberry Pi would control the USB Webcam to take a picture and then send out an alert email with the picture to the user. The advantage of this device is that it is a small size portable stand-alone device with its own power source, it is easy to implement, really cheap for residential use, and energy efficient with instantaneous alert. For the 3D mouse, the MPU6050 would use both the accelerometer and gyroscope to sense user hands movement, the data are processed by MSP430G2553 through a digital smooth filter and a complementary filter, and then the filtered data will pass to the personal computer through the serial COM port. By applying the cursor movement equation in the PC driver, this device can work great as a mouse with acceptable accuracy. Compared to the normal optical mouse we are using, this mouse does not need any working surface, with the use of the smooth and complementary filter, it has certain accuracy for normal use, and it is easy to be extended to a portable mouse as small as a finger ring.

AGENT BASED, DISTRIBUTED CONTROL STRATEGIES AND OPTIMIZATION OF PLUG-IN ELECTRIC VEHICLES ON SMART/MICROGRID ARCHITECTURES

This thesis will present strategies for the use of plug-in electric vehicles on smart and microgrids. MATLAB is used as the design tool for all models and simulations. First, a scenario will be explored using the dispatchable loads of electric vehicles to stabilize a microgrid with a high penetration of renewable power generation. Grid components for a microgrid with 50% photovoltaic solar production will be sized through an optimization routine to maintain storage system, load, and vehicle states over a 24-hour period. The findings of this portion are that the dispatchable loads can be used to guard against unpredictable losses in renewable generation output. Second, the use of distributed control strategies for the charging of electric vehicles utilizing an agent-based approach on a smart grid will be studied. The vehicles are regarded as additional loads to a primary forecasted load and use information transfer with the grid to make their charging decisions. Three lightweight control strategies and their effects on the power grid will be presented. The findings are that the charging behavior and peak loads on the grid can be reduced through the use of distributed control strategies.

Smart Foundry - Informationstechnische Unterstützung der Planung, Konstruktion und Fertigung von Formen für den Metallguss

Die Europäische Gießereiindustrie besteht zu einem großen Teil aus Klein- und Mittelständischen Unternehmen, die in Summe einen bedeutenden Anteil der produzierenden Industrie Europas bilden. Traditionell sind diese Unternehmen lediglich in einem geringen Umfang an Forschungs- und Entwicklungsaktivitäten beteiligt, was vielerorts den Einsatz veralteter Technologie und der damit verbundenen Prozesse zur Folge hat.

Enterprise Integration of Smart Objects using Semantic Service Descriptions

Integrating physical objects (smart objects) and enterprise IT systems is still a labor intensive, mainly manual task done by domain experts. On one hand, enterprise IT backend systems are based on service oriented architectures (SOA) and driven by business rule engines or business process execution engines. Smart objects on the other hand are often programmed at very low levels. In this paper we describe an approach that makes the integration of smart objects with such backends systems easier. We introduce semantic endpoint descriptions based on Linked USDL. Furthermore, we show how different communication patterns can be integrated into these endpoint descriptions. The strength of our endpoint descriptions is that they can be used to automatically create REST or SOAP endpoints for enterprise systems, even if which they are not able to talk to the smart objects directly. We evaluate our proposed solution with CoAP, UDP and 6LoWPAN, as we anticipate the industry converge towards these standards. Nonetheless, our approach also allows easy integration with backend systems, even if no standardized protocol is used.

Smart Classrooms: Guidelines for Educational Technology in Classroom Facility in The University of Texas System

Problem Statement: Classroom facilities developed as new construction or renovation projects for UT System institutions tend to be developed as individual, ad hoc project. There are significant opportunities for process improvement is establishing standard business processes for developing Smart Classroom, establishing design standards and referring to prototype facilities developed at other institutions. [See PDF for complete abstract]

The molecular cloning and characterization of human heparanase cDNA and the immunochemical localization of heparanase in metastatic melanomas

Heparanase, an endo-$\beta$-D-glucuronidase, has been associated with melanoma metastasis. Polyclonal antibodies directed against the murine N-terminal heparanase peptide detected a M$\sb{\rm r}\sim 97,000$ protein upon SDS-polyacrylamide gel electrophoresis of mouse melanoma and human melanoma cell lysates. In an indirect immunocytochemical study, metastatic human A375-SM and mouse B16-BL6 melanoma cells were stained with the anti-heparanase antibodies. Heparanase antigen was localized in the cytoplasm of permeabilized melanoma cells as well as at the cell surface of unpermeabilized cells. Immunohistochemical staining of frozen sections from syngeneic mouse organs containing micrometastases of B16-BL6 melanoma demonstrated heparanase localized in metastatic melanoma cells, but not in adjacent normal tissues. Similar studies using frozen sections of malignant melanomas resected from patients indicated that heparanase is localized in invading melanoma cells, but not in adjacent connective tissues.^ Monoclonal antibodies directed against murine heparanase were developed and characterized. Monoclonal antibody 10E5, an IgM, precipitated and inhibitated the enzymatic activity of heparanase. A 2.6 kb cDNA was isolated from a human melanoma $\lambda$gt11 cDNA library using the monoclonal antibody 10E5. Heparan sulfate cleavage activity was detected in the lysogen lysates from E. Coli Y1089 infected with the $\lambda$gt11 cDNA and this activity was inhibited in the presence of 10-fold excess of heparin, a potent inhibitor of heparanase. The nucleotide sequence of the cDNA was determined and insignificant homology was found with the gene sequences currently known. The cDNA hybridized to a 3.2-3.4 kb mRNA in human A375 melanoma, WI-38 fibroblast, and THP-1 leukemia cells using Northern blots.^ Heparanase expression was examined using Western and Northern blots. In comparison to human A375-P melanoma cells, the quantity of 97,000 protein recognized by the polyclonal anti-heparanase antibodies doubled in the metastatic variant A375-SM cells and the quantity of 3.2-3.4 kb mRNA doubled in A375MetMix, a metastatic variant similar to A375-SM cells. In B16 murine melanoma cell, the intensity of the 97,000 protein increased more than 2 times comparing with B16-F1 cells. The extent in the increase of the protein and the mRNA levels is comparable to the change of heparanase activity observed in those cells.^ In summary, the studies suggest that (a) the N-terminus of the heparanase molecule in mouse and human is antigenically related; (b) heparanase antigens are localized at the cell surface and in the cytoplasm of metastatic human and mouse melanoma cells; (c) heparanase antigens are localized in invasive and metastatic murine and human melanomas in vivo, but not in adjacent normal tissues; (d) heparanase molecule appeared to be differentially expressed at the transcriptional as well as at the translational level; and (e) the size of human heparanase mRNA is 3.2-3.4 kilobase. ^

cDNA cloning and expression of a novel cell surface-expressed \b heparan sulfate/heparin \b interacting \b protein (HIP) from human cell lines and functional studies of a synthetic HIP peptide

Heparan sulfate proteoglycans and their corresponding binding sites have been suggested to play an important role during the initial attachment of blastocysts to uterine epithelium and human trophoblastic cell lines to uterine epithelial cell lines. Previous studies on RL95 cells, a human uterine epithelial cell line, characterized a single class of cell surface heparin/heparan sulfate (HP/HS)-binding sites. Three major HP/HS-binding peptide fragments were isolated from RL95 cell surfaces by tryptic digestion and partial amino-terminal amino acid sequence from each peptide fragment was obtained. In the current study, using the approaches of reverse transcription-polymerase chain reaction and cDNA library screening, a novel cell surface $\rm\underline{H}$P/HS $\rm\underline{i}$nteracting $\rm\underline{p}$rotein (HIP) has been isolated from RL95 cells. The full-length cDNA of HIP encodes a protein of 259 amino acids with a calculated molecular weight of 17,754 Da and pI of 11.75. Transfection of HIP cDNA into NIH-3T3 cells demonstrated cell surface expression and a size similar to that of HIP expressed by human cells. Predicted amino acid sequence indicates that HIP lacks a membrane spanning region and has no consensus sites for glycosylation. Northern blot analysis detected a single transcript of 1.3 kb in both total RNA and poly(A$\sp+$) RNA. Examination of human cell lines and normal tissues using both Northern blot and Western blot analysis revealed that HIP is differentially expressed in a variety of human cell lines and normal tissues, but absent in some cell lines examined. HIP has about 80% homology, at the level of both mRNA and protein, to a rodent protein, designated as ribosomal protein L29. Thus, members of the L29 family may be displayed on cell surfaces where they participate in HP/HS binding events. Studies on a synthetic peptide derived from HIP demonstrate that HIP peptide binds HS/HP with high selectivity and has high affinity (Kd = 10 nM) for a subset of polysaccharides found in commercial HIP preparations. Moreover, HIP peptide also binds certain forms of cell surface, but not secreted or intracellular. HS expressed by RL95 and JAR cells. This peptide supports the attachment of several human trophoblastic cell lines and a variety of mammalian adherent cell lines in a HS-dependent fashion. Furthermore, studies on the subset of HP specifically recognized by HIP peptide indicate that this high-affinity HP (HA-HP) has a larger median MW and a greater negative charge density than bulk HP. The minimum size of oligosaccharide required to bind to HIP peptide with high affinity is a septa- or octasaccharide. HA-HP also quantitatively binds to antithrombin-III (AT-III) with high affinity, indicating that HIP peptide and AT-III may recognize the same or similar oligosaccharide structure(s). Furthermore, HIP peptide antagonizes HP action and promotes blood coagulation in both factor Xa- and thrombin-dependent assays. Finally, HA-HP recognized by HP peptide is highly enriched with anticoagulant activity relative to bulk HP. Collectively, these results demonstrate that HIP may play a role in the HP/HS-involved cell-cell and cell-matrix interactions and recognizes a motif in HP similar or identical to that recognized by AT-III and therefore, may modulate blood coagulation. ^

The Integration of Electricity from Renewable Energy Sources in the European Union Electricity Market – The case for “Smart Grids”

The power sector is to play a central role in a low carbon economy. In all the decarbonisation scenarios of the European Union renewable energy sources (RES) will be a crucial part of the solution. Current grids constitute however major bottlenecks for the future expansion of RES. Recognising the need for a modernisation of its grids, the European Union has called for the creation of a "smart supergrid" interconnecting European grids at the continental level and making them "intelligent" through the addition of information and communication technology (ICT). To implement its agenda the EU has taken a leading role in coordinating research efforts and creating a common legislative framework for the necessary modernisation of Europe’s grids. This paper intends to give both an overview and a critical appraisal of the measures taken so far by the European Union to "transform" the grids into the backbone of a decarbonised electricity system. It suggests that if competition is to play a significant role in the deployment of smart grids, the current regulatory paradigm will have to be fundamentally reassessed

A novel form of CYP3A from rat brain: cDNA cloning, protein characterization and gene regulation of cytochrome P450 3A9

One full length cDNA clone, designated 3aH15, was isolated from a rat brain cDNA library using a fragment of CYP3A2 cDNA as a probe. 3aH15 encoded a protein composed of 503 amino acid residues. The deduced amino acid sequence of 3aH15 was 92% identical to mouse Cyp3a-13 and had a 68.4% to 76.5% homology with the other reported rat CYP3A sequences. Clone 3aH15 was thus named CYP3A9 by Cytochrome P450 Nomenclature Committee. CYP3A9 seems to the major CYP3A isozyme expressed in rat brain. Sexual dimorphism of the expression of CYP3A9 was shown for the first time in rat brain as well as in rat liver. CYP3A9 appears to be female specific in rat liver based on the standards proposed by Kato and Yamazoe who defined sex specific expression of P450s as being a 10-fold or higher expression level in one sex compared with the other. CYP3A9 gene expression was inducible by estrogen treatment both in male and in female rats. Male rats treated with estrogen had a similar expression level of CYP3A9 mRNA both in the liver and brain. Ovariectomy of adult female rats drastically reduced the mRNA level of CYP3A9 which could be fully restored by estrogen replacement. On the other hand, only a two-fold induction of CYP3A9 expression by dexamethasone was observed in male liver and no significant induction of CYP3A9 mRNA was observed in female liver or in the brains. These results suggest that estrogen may play an important role in the female specific expression of the CYP3A9 gene and that CYP3A9 gene expression is regulated differently from other CYP3A isozymes. ^ P450 3A9 recombinant protein was expressed in E. coli using the pCWOri+ expression vector and the MALLLAVF amino terminal sequence modification. This construct gave a high level of expression (130 nmol P450 3A9/liter culture) and the recombinant protein of the modified P450 3A9 was purified to electrophoretic homogeneity (10.1 nmol P450/mg protein) from solubilized fractions using two chromatographic steps. The purified P450 3A9 protein was active towards the metabolism of many clinically important drugs such as imipramine, erythromycin, benzphetamine, ethylmorphine, chlorzoxazone, cyclosporine, rapamycin, etc. in a reconstituted system containing lipid and rat NADPH-P450 reductase. Although P450 3A9 was active towards the catabolism of testosterone, androstenedione, dehydroepiandrosterone (DHEA) and 17β-estradiol, P450 3A9 preferentially catalyzes the metabolism of progesterone to form four different hydroxylated products. Optimal reconstitution conditions for P450 3A9 activities required a lipid mixture and GSH. The possible mechanisms of the stimulatory effects of GSH on P450 3A9 activities are discussed. Sexually dimorphic expression of P450 3A9 in the brain and its involvement in many neuroactive drugs as well as neurosteroids suggest the possible role of P450 3A9 in some mental disorders and brain functions. ^

Smart Cities – Ein Überblick!

Nach einer kurzen Begriffsfassung von Smart Cities gehen wir basierend auf den folgenden Beiträgen dieses Heftes auf verschiedene Eigenschaften einer solchen smarten Stadt ein. Dadurch versuchen wir den Ist-Zustand dieser Städte zu dokumentieren. Damit die jeweiligen Stakeholder (strategische) Entscheide treffen können, widmen wir danach ein Kapitel den Chancen und Risiken von Smart Cities. Anhand einer Studie des Europäischen Parlaments zeigen wir nachfolgend entsprechende Bestrebungen aus Europa auf. Anschliessend präsentieren wir eine Best-Practice-Roadmap für die Realisierung von Smart Cities. Zum Schluss zeichnen wir auf einer konnektivistischen Lern- und Kognitionstheorie aufbauend einen Weg zur Cognitive City der Zukunft. Dabei wird der Mensch nicht als isoliertes, sondern als vernetztes Individuum gesehen. Dies begünstigt die Weiterentwicklung von Smart Cities zu Städten, welche aktiv und selbstständig lernen und dadurch automatisch auf Veränderungen ihrer Umwelt reagieren können.

Comprehensive measures of sound exposures in cinemas using smart phones

OBJECTIVES Sensorineural hearing loss from sound overexposure has a considerable prevalence. Identification of sound hazards is crucial, as prevention, due to a lack of definitive therapies, is the sole alternative to hearing aids. One subjectively loud, yet little studied, potential sound hazard is movie theaters. This study uses smart phones to evaluate their applicability as a widely available, validated sound pressure level (SPL) meter. Therefore, this study measures sound levels in movie theaters to determine whether sound levels exceed safe occupational noise exposure limits and whether sound levels in movie theaters differ as a function of movie, movie theater, presentation time, and seat location within the theater. DESIGN Six smart phones with an SPL meter software application were calibrated with a precision SPL meter and validated as an SPL meter. Additionally, three different smart phone generations were measured in comparison to an integrating SPL meter. Two different movies, an action movie and a children's movie, were measured six times each in 10 different venues (n = 117). To maximize representativeness, movies were selected focusing on large release productions with probable high attendance. Movie theaters were selected in the San Francisco, CA, area based on whether they screened both chosen movies and to represent the largest variety of theater proprietors. Measurements were analyzed in regard to differences between theaters, location within the theater, movie, as well as presentation time and day as indirect indicator of film attendance. RESULTS The smart phone measurements demonstrated high accuracy and reliability. Overall, sound levels in movie theaters do not exceed safe exposure limits by occupational standards. Sound levels vary significantly across theaters and demonstrated statistically significant higher sound levels and exposures in the action movie compared to the children's movie. Sound levels decrease with distance from the screen. However, no influence on time of day or day of the week as indirect indicator of film attendance could be found. CONCLUSIONS Calibrated smart phones with an appropriate software application as used in this study can be utilized as a validated SPL meter. Because of the wide availability, smart phones in combination with the software application can provide high quantity recreational sound exposure measurements, which can facilitate the identification of potential noise hazards. Sound levels in movie theaters decrease with distance to the screen, but do not exceed safe occupational noise exposure limits. Additionally, there are significant differences in sound levels across movie theaters and movies, but not in presentation time.