Subjective QoE Analysis of HTTP Adaptive Streaming Applications

Actualmente la optimization de la calidad de experiencia (Quality of Experience- QoE) de HTTP Adaptive Streaming (HAS) de video recibe una atención creciente. Este incremento de interés proviene fundamentalmente de las carencias de las soluciones actuales HAS, que, al no ser QoE-driven, no incluyen la percepción de la calidad de los usuarios finales como una parte integral de la lógica de adaptación. Por lo tanto, la obtención de información de referencia fiable en QoE en HAS presenta retos importantes, ya que las metodologías de evaluación subjetiva de la calidad de vídeo propuestas en las normas actuales no son adecuadas para tratar con la variación temporal de la calidad que es consustancial de HAS. Esta tesis investiga la influencia de la adaptación dinámica en la calidad de la transmisión de vídeo considerando métodos de evaluación subjetiva. Tras un estudio exhaustivo del estado del arte en la evaluación subjetiva de QoE en HAS, se han resaltado los retos asociados y las líneas de investigación abiertas. Como resultado, se han seleccionado dos líneas principales de investigación: el análisis del impacto en la QoE de los parámetros de las técnicas de adaptación y la investigación de las metodologías de prueba subjetiva adecuada para evaluación de QoE en HAS. Se han llevado a cabo un conjunto de experimentos de laboratorio para investigar las cuestiones planteadas mediante la utilización de diferentes metodologáas para pruebas subjetivas. El análisis estadístico muestra que no son robustas todas las suposiciones y reivindicaciones de las referencias analizadas, en particular en lo que respecta al impacto en la QoE de la frecuencia de las variaciones de calidad, de las adaptaciones suaves o abruptas y de las oscilaciones de calidad. Por otra parte, nuestros resultados confirman la influencia de otros parámetros, como la longitud de los segmentos de vídeo y la amplitud de las oscilaciones de calidad. Los resultados también muestran que tomar en consideración las características objetivas de los contenidos puede ser beneficioso para la mejora de la QoE en HAS. Además, todos los resultados han sido validados mediante extensos análisis experimentales que han incluido estudio tanto en otros laboratorios como en crowdsourcing Por último, sobre los aspectos metodológicos de las pruebas subjetivas de QoE, se ha realizado la comparación entre los resultados experimentales obtenidos a partir de un método estandarizado basado en estímulos cortos (ACR) y un método semi continuo (desarrollado para la evaluación de secuencias prolongadas de vídeo). A pesar de algunas diferencias, el resultado de los análisis estadísticos no muestra ningún efecto significativo de la metodología de prueba. Asimismo, aunque se percibe la influencia de la presencia de audio en la evaluación de degradaciones del vídeo, no se han encontrado efectos estadísticamente significativos de dicha presencia. A partir de la ausencia de influencia del método de prueba y de la presencia de audio, se ha realizado un análisis adicional sobre el impacto de realizar comparaciones estadísticas múltiples en niveles estadísticos de importancia que aumentan la probabilidad de los errores de tipo-I (falsos positivos). Nuestros resultados muestran que, para obtener un efectos sólido en el análisis estadístico de los resultados subjetivos, es necesario aumentar el número de sujetos de las pruebas claramente por encima de los tamaños de muestras propuestos por las normas y recomendaciones actuales. ABSTRACT Optimizing the Quality of Experience (QoE) of HTTP adaptive video streaming (HAS) is receiving increasing attention nowadays. The growth of interest is mainly caused by the fact that current HAS solutions are not QoE-driven, i.e. end-user quality perception is not integral part of the adaptation logic. However, obtaining the necessary reliable ground truths on HAS QoE faces substantial challenges, since the subjective video quality assessment methodologies as proposed by current standards are not well-suited for dealing with the time-varying quality properties that are characteristic for HAS. This thesis investigates the influence of dynamic quality adaptation on the QoE of streaming video by means of subjective evaluation approaches. Based on a comprehensive survey of related work on subjective HAS QoE assessment, the related challenges and open research questions are highlighted and discussed. As a result, two main research directions are selected for further investigation: analysis of the QoE impact of different technical adaptation parameters, and investigation of testing methodologies suitable for HAS QoE evaluation. In order to investigate related research issues and questions, a set of laboratory experiments have been conducted using different subjective testing methodologies. Our statistical analysis demonstrates that not all assumptions and claims reported in the literature are robust, particularly as regards the QoE impact of switching frequency, smooth vs. abrupt switching, and quality oscillation. On the other hand, our results confirm the influence of some other parameters such as chunk length and switching amplitude on perceived quality. We also show that taking the objective characteristics of the content into account can be beneficial to improve the adaptation viewing experience. In addition, all aforementioned findings are validated by means of an extensive cross-experimental analysis that involves external laboratory and crowdsourcing studies. Finally, to address the methodological aspects of subjective QoE testing, a comparison between the experimental results obtained from a (short stimuli-based) ACR standardized method and a semi-continuous method (developed for assessment of long video sequences) has been performed. In spite of observation of some differences, the result of statistical analysis does not show any significant effect of testing methodology. Similarly, although the influence of audio presence on evaluation of video-related degradations is perceived, no statistically significant effect of audio presence could be found. Motivating by this finding (no effect of testing method and audio presence), a subsequent analysis has been performed investigating the impact of performing multiple statistical comparisons on statistical levels of significance which increase the likelihood of Type-I errors (false positives). Our results show that in order to obtain a strong effect from the statistical analysis of the subjective results, it is necessary to increase the number of test subjects well beyond the sample sizes proposed by current quality assessment standards and recommendations.

Quality of experience of adaptive video streaming: investigation in service parameters and subjective quality assessment methodology

The usage of HTTP adaptive streaming (HAS) has become widely spread in multimedia services. Because it allows the service providers to improve the network resource utilization and user׳s Quality of Experience (QoE). Using this technology, the video playback interruption is reduced since the network and server status in addition to capability of user device, all are taken into account by HAS client to adapt the quality to the current condition. Adaptation can be done using different strategies. In order to provide optimal QoE, the perceptual impact of adaptation strategies from point of view of the user should be studied. However, the time-varying video quality due to the adaptation which usually takes place in a long interval introduces a new type of impairment making the subjective evaluation of adaptive streaming system challenging. The contribution of this paper is two-fold: first, it investigates the testing methodology to evaluate HAS QoE by comparing the subjective experimental outcomes obtained from ACR standardized method and a semi-continuous method developed to evaluate the long sequences. In addition, influence of using audiovisual stimuli to evaluate the video-related impairment is inquired. Second, impact of some of the adaptation technical factors including the quality switching amplitude and chunk size in combination with high range of commercial content type is investigated. The results of this study provide a good insight toward achieving appropriate testing method to evaluate HAS QoE, in addition to designing switching strategies with optimal visual quality.

Caracterização geométrica e mecânica de macrofibras poliméricas.

Uma grande diversidade de macrofibras poliméricas para reforço de concreto se encontram disponibilizadas hoje em dia. Por natureza estas fibras apresentam grande diversidade de características e propriedades. Estas variações afetam sua atuação como reforço no concreto. No entanto, não há normas brasileiras sobre o assunto e as metodologias de caracterização de normas estrangeiras apresentam divergências. Algumas normas definem que a caracterização do comportamento mecânico deva ser feita nos fios originais e outras que se devam utilizar métodos definidos para caracterização de materiais metálicos. A norma EN14889-2:2006 apresenta maior abrangência, mas deixa dúvidas quanto à adequação dos critérios de caracterização geométrica das fibras e não define um método de ensaio específico para sua caracterização mecânica. Assim, há a necessidade de estabelecimento de uma metodologia que permita a realização de um programa de controle de qualidade da fibra nas condições de emprego. Esta metodologia também proporcionaria uma forma de caracterização do material para estudos experimentais, o que permitiria maior fundamentação científica desses trabalhos que, frequentemente, fundamentam-se apenas em dados dos fabricantes. Assim, foi desenvolvido um estudo experimental focando a caracterização de duas macrofibras poliméricas disponíveis no mercado brasileiro. Focou-se o estudo na determinação dos parâmetros geométricos e na caracterização mecânica através da determinação da resistência à tração e avaliação do módulo de elasticidade. Na caracterização geométrica foi adotada como referência a norma europeia EN14889-2:2006. As medições do comprimento se efetuaram por dois métodos: o método do paquímetro e o método de análise de imagens digitais, empregando um software para processamento das imagens. Para a medição do diâmetro, além das metodologias mencionadas, foi usado o método da densidade. Conclui-se que o método do paquímetro, com o cuidado de esticar previamente as macrofibras, e o método das imagens digitais podem ser igualmente utilizados para medir o comprimento. Já parar determinar o diâmetro, recomenda-se o método da densidade. Quanto à caracterização mecânica, foi desenvolvida uma metodologia própria a partir de informações obtidas de outros ensaios. Assim, efetuaram-se ensaios de tração direta nas macrofibras coladas em molduras de tecido têxtil. Complementarmente, foi avaliado também o efeito do contato abrasivo das macrofibras com os agregados durante a mistura em betoneira no comportamento mecânico do material. Também se avaliou o efeito do método de determinação da área da seção transversal nos resultados medidos no ensaio de tração da fibra. Conclui-se que o método proposto para o ensaio de tração direta da fibra é viável, especialmente para a determinação da resistência à tração. O valor do módulo de elasticidade, por sua vez, acaba sendo subestimado. A determinação da área da seção da fibra através do método da densidade forneceu também os melhores resultados. Além disso, comprovou-se que o atrito das fibras com o agregado durante a mistura compromete o comportamento mecânico, reduzindo tanto a resistência quanto o módulo de elasticidade. Assim, pode-se afirmar que a metodologia proposta para o controle geométrico e mecânico das macrofibras poliméricas é adequada para a caracterização do material.

Mechanical properties of interfacial films formed by lysozyme self-assembly at the air-water interface

We present the first characterization of the mechanical properties of lysozyme films formed by self-assembly at the air-water interface using the Cambridge interfacial tensiometer (CIT), an apparatus capable of subjecting protein films to a much higher level of extensional strain than traditional dilatational techniques. CIT analysis, which is insensitive to surface pressure, provides a direct measure of the extensional stress-strain behavior of an interfacial film without the need to assume a mechanical model (e.g., viscoelastic), and without requiring difficult-to-test assumptions regarding low-strain material linearity. This testing method has revealed that the bulk solution pH from which assembly of an interfacial lysozyme film occurs influences the mechanical properties of the film more significantly than is suggested by the observed differences in elastic moduli or surface pressure. We have also identified a previously undescribed pH dependency in the effect of solution ionic strength on the mechanical strength of the lysozyme films formed at the air-water interface. Increasing solution ionic strength was found to increase lysozyme film strength when assembly occurred at pH 7, but it caused a decrease in film strength at pH 11, close to the pI of lysozyme. This result is discussed in terms of the significant contribution made to protein film strength by both electrostatic interactions and the hydrophobic effect. Washout experiments to remove protein from the bulk phase have shown that a small percentage of the interfacially adsorbed lysozyme molecules are reversibly adsorbed. Finally, the washout tests have probed the role played by additional adsorption to the fresh interface formed by the application of a large strain to the lysozyme film and have suggested the movement of reversibly bound lysozyme molecules from a subinterfacial layer to the interface.

Development and evaluation of a non-destructive adhesion test

The object of this work was to further develop the idea introduced by Muaddi et al (1981) which enables some of the disadvantages of earlier destructive adhesion test methods to be overcome. The test is non-destructive in nature but it does need to be calibrated against a destructive method. Adhesion is determined by measuring the effect of plating on internal friction. This is achieved by determining the damping of vibrations of a resonating specimen before and after plating. The level of adhesion was considered by the above authors to influence the degree of damping. In the major portion of the research work the electrodeposited metal was Watt's nickel, which is ductile in nature and is therefore suitable for peel adhesion testing. The base metals chosen were aluminium alloys S1C and HE9 as it is relatively easy to produce varying levels of adhesion between the substrate and electrodeposited coating by choosing the appropriate process sequence. S1C alloy is the commercially pure aluminium and was used to produce good adhesion. HE9 aluminium alloy is a more difficult to plate alloy and was chosen to produce poorer adhesion. The "Modal Testing" method used for studying vibrations was investigated as a possible means of evaluating adhesion but was not successful and so research was concentrated on the "Q" meter. The method based on the use of a "Q" meter involves the principle of exciting vibrations in a sample, interrupting the driving signal and counting the number of oscillations of the freely decaying vibrations between two known preselected amplitudes of oscillations. It was not possible to reconstruct a working instrument using Muaddi's thesis (1982) as it had either a serious error or the information was incomplete. Hence a modified "Q" meter had to be designed and constructed but it was then difficult to resonate non-magnetic materials, such as aluminium, therefore, a comparison before and after plating could not be made. A new "Q" meter was then developed based on an Impulse Technique. A regulated miniature hammer was used to excite the test piece at the fundamental mode instead of an electronic hammer and test pieces were supported at the two predetermined nodal points using nylon threads. This instrument developed was not very successful at detecting changes due to good and poor pretreatments given before plating, however, it was more sensitive to changes at the surface such as room temperature oxidation. Statistical analysis of test results from untreated aluminium alloys show that the instrument is not always consistent, the variation was even bigger when readings were taken on different days. Although aluminium is said to form protective oxides at room temperature there was evidence that the aluminium surface changes continuously due to film formation, growth and breakdown. Nickel plated and zinc alloy immersion coated samples also showed variation in Q with time. In order to prove that the variations in Q were mainly due to surface oxidation, aluminium samples were lacquered and anodised Such treatments enveloped the active surfaces reacting with the environment and the Q variation with time was almost eliminated especially after hard anodising. This instrument detected major differences between different untreated aluminium substrates.Also Q values decreased progressively as coating thicknesses were increased. This instrument was also able to detect changes in Q due to heat-treatment of aluminium alloys.

A sulphonate based hydrogel for nucleus pulposus repair

Introduction: Lower back pain treatment and compensation costs >$80 billion overall in the US. 75% of back pain is due to disc degeneration in the lumbar region of the spine. Current treatment comprises of painkillers and bed rest or as a more radical solution – interbody cage fusion. In the early stages of disc degeneration the patient would benefit from addition of an injectable gel which polymerises in situ to support the degenerated nucleus pulposus. This involves a material which is an analogue of the natural tissue capable of restoring the biomechanical properties of the natural disc. The nucleus pulposus of the intervertebral disc is an example of a natural proteoglycan consisting of a protein core with negatively charged keratin and chondroitin sulphate attached. As a result of the high fixed charge density of the proteoglycan, the matrix exerts an osmotic swelling pressure drawing sufficient water into support the spinal system. Materials and Methods: NaAMPs (sodium 2- acrylamido 2-methyl propane sulphonic acid) and KSPA (potassium 3- sulphopropyl acrylate) were selected as monomers, the sulphonate group being used to mimic the natural sulphate group. These are used in dermal applications involving chronic wounds and have acceptably low cytotoxicity. Other hydrophilic carboxyl, amide and hydroxyl monomers such as 2-hydroxyethyl acrylamide, ß-carboxyethyl acrylate, acryloyl morpholine, and polyethylene glycol (meth)acrylate were used as diluents together with polyethyleneglycol di(meth)acrylate and hydrophilic multifunctional macromers as cross-linker. Redox was the chosen method of polymerisation and a range of initiators were investigated. Components were packaged in two solutions each containing a redox pair. A dual syringe method of injection into the cavity was used, the required time for polymerisation is circa 3-7 minutes. The final materials were tested using a Bohlin CVO Rheometer cycling from 0.5-25Hz at 37oC to measure the modulus. An in-house compression testing method was developed, using dialysis tubing to mimic the cavity, the gels were swelled in solutions of various osmolarity and compressed to ~ 20%. The pre-gel has also been injected into sheep spinal segments for mechanical compression testing to demonstrate the restoration of properties upon use of the gel. Results and Discussion: Two systems resulted using similar monomer compositions but different initiation and crosslinking agents. NaAMPs and KSPA were used together at a ratio of ~1:1 in both systems with 0.25-2% crosslinking agent, diacrylate or methacrylate. The two initiation systems were ascorbic acid/oxone, and N,N,N,N - tetramethylethylenediamine (TEMED)/ potassium persulphate. These systems produced gelation within 3-7 and 3-5 minutes respectively. Storage of the two component systems was shown to be stable for approximately one month after mixing, in the dark, refrigerated at 1-4oC. The gelation was carried out at 37oC. Literature values for the natural disc give elastic constants ranging from 3-8kPa. The properties of the polymer can be tailored by altering crosslink density and monomer composition and are able to match those of the natural disc. It is possible to incorporate a radio-opaque (histodenz) to enable x-ray luminescence during and after injection. At an inclusion level of 5% the gel is clearly visible and polymerisation and mechanical properties are not altered. Conclusion: A two-pac injection system which will polymerise in situ, that can incorporate a radio-opaque, has been developed. This will reinforce the damaged nucleus pulposus in degenerative disc disease restoring adequate hydration and thus biomechanical properties. Tests on sheep spine segments are currently being carried out to demonstrate that a disc containing the gel has similar properties to an intact disc in comparison to one with a damaged nucleus.

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Finite Element Modeling of Spiral Frequency Steerable Acoustic Transducers (FSATs) for guided waves based Structural Health Monitoring of plate-like structures

Structural Health Monitoring (SHM) is an emerging area of research associated to improvement of maintainability and the safety of aerospace, civil and mechanical infrastructures by means of monitoring and damage detection. Guided wave structural testing method is an approach for health monitoring of plate-like structures using smart material piezoelectric transducers. Among many kinds of transducers, the ones that have beam steering feature can perform more accurate surface interrogation. A frequency steerable acoustic transducer (FSATs) is capable of beam steering by varying the input frequency and consequently can detect and localize damage in structures. Guided wave inspection is typically performed through phased arrays which feature a large number of piezoelectric transducers, complexity and limitations. To overcome the weight penalty, the complex circuity and maintenance concern associated with wiring a large number of transducers, new FSATs are proposed that present inherent directional capabilities when generating and sensing elastic waves. The first generation of Spiral FSAT has two main limitations. First, waves are excited or sensed in one direction and in the opposite one (180 ̊ ambiguity) and second, just a relatively rude approximation of the desired directivity has been attained. Second generation of Spiral FSAT is proposed to overcome the first generation limitations. The importance of simulation tools becomes higher when a new idea is proposed and starts to be developed. The shaped transducer concept, especially the second generation of spiral FSAT is a novel idea in guided waves based of Structural Health Monitoring systems, hence finding a simulation tool is a necessity to develop various design aspects of this innovative transducer. In this work, the numerical simulation of the 1st and 2nd generations of Spiral FSAT has been conducted to prove the directional capability of excited guided waves through a plate-like structure.

Processamento e caracterização da blenda poli (metacrilato de metila) (PMMA) elastomerico e poli (tereftalato de etileno) (PET) pós-consumo

This work studied the immiscible blend of elastomeric poly(methyl methacrylate) (PMMA) with poly(ethylene terephthalate) (PET) bottle grade with and without the use of compatibilizer agent, poly(methyl methacrylate-co-glycidyl methacrylate - co-ethyl acrylate) (MGE). The characterizations of torque rheometry, melt flow index measurement (MFI), measuring the density and the degree of cristallinity by pycnometry, tensile testing, method of work essential fracture (EWF), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed in pure polymer and blends PMMA/PET. The rheological results showed evidence of signs of chemical reaction between the epoxy group MGE with the end groups of the PET chains and also to the elastomeric phase of PMMA. The increase in the concentration of PET reduced torque and adding MGE increased the torque of the blend of PMMA/PET. The results of the MFI also show that elastomeric PMMA showed lower flow and thus higher viscosity than PET. In the results of picnometry observed that increasing the percentage of PET resulted in an increase in density and degree crystallinity of the blends PMMA/PET. The tensile test showed that increasing the percentage of PET resulted in an increase in ultimate strength and elastic modulus and decrease in elongation at break. However, in the phase inversion, where the blend showed evidence of a co-continuous morphology and also, with 30% PET dispersed phase and compatibilized with 5% MGE, there were significant results elongation at break compared to elastomeric PMMA. The applicability of the method of essential work of fracture was shown to be possible for most formulations. And it was observed that with increasing elastomeric PMMA in the formulations of the blends there was an improvement in specific amounts of essential work of fracture (We) and a decrease in the values of specific non-essential work of fracture (βWp)

In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic adenovirus

Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.

An analytical method for assessing stage-specific drug activity in Plasmodium vivax malaria : implications for ex vivo drug susceptibility testing

The emergence of highly chloroquine (CQ) resistant P. vivax in Southeast Asia has created an urgent need for an improved understanding of the mechanisms of drug resistance in these parasites, the development of robust tools for defining the spread of resistance, and the discovery of new antimalarial agents. The ex vivo Schizont Maturation Test (SMT), originally developed for the study of P. falciparum, has been modified for P. vivax. We retrospectively analysed the results from 760 parasite isolates assessed by the modified SMT to investigate the relationship between parasite growth dynamics and parasite susceptibility to antimalarial drugs. Previous observations of the stage-specific activity of CQ against P. vivax were confirmed, and shown to have profound consequences for interpretation of the assay. Using a nonlinear model we show increased duration of the assay and a higher proportion of ring stages in the initial blood sample were associated with decreased effective concentration (EC50) values of CQ, and identify a threshold where these associations no longer hold. Thus, starting composition of parasites in the SMT and duration of the assay can have a profound effect on the calculated EC50 for CQ. Our findings indicate that EC50 values from assays with a duration less than 34 hours do not truly reflect the sensitivity of the parasite to CQ, nor an assay where the proportion of ring stage parasites at the start of the assay does not exceed 66%. Application of this threshold modelling approach suggests that similar issues may occur for susceptibility testing of amodiaquine and mefloquine. The statistical methodology which has been developed also provides a novel means of detecting stage-specific drug activity for new antimalarials.

Cytotoxicity testing of burn wound dressings, ointments and creams : a method using polycarbonate cell culture inserts on a cell culture system

We have developed a method to test the cytotoxicity of wound dressings, ointments, creams and gels used in our Burn Centre, by placing them on a permeable Nunc Polycarbonate cell culture insert, incubated with a monolayer of cells (HaCaTs and primary human keratinocytes). METHODS: We performed two different methods to determine the relative toxicity to cells. (1) Photo visualisation: The dressings or compounds were positioned on the insert's membrane which was placed onto the monolayer tissue culture plate. After 24 h the surviving adherent cells were stained with Toluidine Blue and photos of the plates were taken. The acellular area of non-adherent dead cells which had been washed off with buffer was measured as a percentage of the total area of the plate. (2) Cell count of surviving cells: After 24 h incubation with the test material, the remaining cells were detached with trypsin, spun down and counted in a Haemocytometer with Trypan Blue, which differentiates between live and dead cells. RESULTS: Seventeen products were tested. The least cytotoxic products were Melolite, White soft Paraffin and Chlorsig1% Ointment. Some cytotoxicity was shown with Jelonet, Mepitel((R)), PolyMem((R)), DuoDerm((R)) and Xeroform. The most cytotoxic products included those which contained silver or Chlorhexidine and Paraffin Cream a moisturizer which contains the preservative Chlorocresol. CONCLUSION: This in vitro cell culture insert method allows testing of agents without direct cell contact. It is easy and quick to perform, and should help the clinician to determine the relative cytotoxicity of various dressings and the optimal dressing for each individual wound.

Rapid method of consolidation testing

The determination of consolidation characteristics forms an important aspect in the design of foundations and other earth-retaining structures. The conventional consolidation test as originally proposed by Teaaghi takes considerable time (more than 15 days in highly compressible soils with low coefficient of consolidation) and effort. Any effort to reduce the duration of testing will be desirable from several considerations. In this paper, an attempt has been made to propose a rapid method of consolidation testing. In the proposed method, the next load increment is applied as soon as the necessary time required to identify the percent consolidation is reached and to evaluate the coefficient of consolidation by one of the popular curve-fitting procedures. The rectangular hyperbola method has been used to identify the percent consolidation reached after any load increment, and to determine the coefficient of consolidation, before making the next load increment. The time required to complete the test using the rapid consolidation method could be as low as 4-5 h compared with 1 or 2 weeks in the case of the conventional consolidation test.