Evaluation of PCR in the molecular diagnosis of endocarditis

Objective. Infective endocarditis (IE) is diagnosed by the Duke criteria, which can be inconclusive particularly when blood cultures are negative. This study investigated the application of polymerase chain reaction (PCR) to identify bacterial DNA in excised valvular tissue, and its role in establishing the diagnosis of IE. Methods. Ninety-eight patients undergoing valve replacement surgery were studied. Twenty-eight patients were confirmed as definite for endocarditis by the Duke criteria; nine were considered as possible and 61 had no known or previous microbial infection of the endocardium. A broad-range PCR technique was used to amplify prokaryotic 16S rRNA genes present within homogenised heart valve tissue. Subsequent DNA sequencing of the PCR amplicon allowed identification of the infecting microorganism. Results. PCR results demonstrated the presence of bacterial DNA in the heart valves obtained from 14 out of 20 (70%) definite IE patients with positive blood cultures preoperatively. The causative microorganism for one patient with definite culture negative endocarditis was identified by PCR. Two out of nine (22%) of the valves from possible endocarditis patients also had bacterial DNA present converting them into the definite criteria whereas in the valves of seven out of nine (78%) of these patients no bacterial DNA was detected. Conclusion. The application of PCR to the explanted valves in patients with possible or confirmed diagnosis can augment the Duke criteria thereby improving post-surgical antimicrobial therapeutic options. © 2003 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

RT-PCR for the pseudogene-free amplification of the glyceraldehyde-3-phosphate dehydrogenase gene (gapd)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme which catalyses the conversion of glyceraldehyde-3-phosphate to 1,3 diphosphoglycerate. It is considered to be constitutively expressed in all cells, and as such the gene for GAPDH (gapd) is commonly used as a benchmark reference in expression studies. However, previous investigations have demonstrated that gapd may show altered gene expression in a number of disease states and under certain experimental conditions, suggesting that results of experiments using gapd as a control should be interpreted with caution. Furthermore, consideration must be given to the potential co-amplification of pseudogenes of gapd during RT-PCR. Here, we describe a method to avoid the amplification of contaminating pseudogenes through the design of primers that bind only to genuine gapd mRNA transcript. © 2003 Elsevier Ltd. All rights reserved.

Detection of self-complementary inverted repeats by single forward primer driven PCR

Inverted repeat gene structures designed for silencing functional genes have been widely used both in academic and applied research. The correct orientations of such structures are usually validated with restriction analysis and/or sequencing. We speculated that the inverted repeat nature of such constructs can be shown by a simple PCR reaction with a single forward primer. To test this hypothesis five different constructs were established from grapevine sequences in a hairpin-intron style silencing system. We were able to amplify the appropriate products in each case. Thus a forward-primed PCR alone may be sufficient to prove the inverted repeat nature of the desired constructs.

Establishment of a PCR and restriction enzyme assay for the detection of the hemochromatosis gene in normal subjects and diabetic patients

The purpose of this study was to establish a polymerase chain reaction (PCR)-restriction enzyme assay for detecting the hereditary hemochromatosis (HHC) mutation, C282Y, in gestational and gestational diabetic subjects in South Florida. DNA samples from 43 gestational subjects were amplified by PCR, digested with RsaI, and analyzed by electrophoresis. An allelic frequency of 2.33%, or 4.65% heterozygosity, was observed. The assay is successful and applicable to future studies on HHC and gestational diabetes. ^

The effects of using rap music on the fluency skills of 2nd grade African American students

A concern for both educators and policy makers is how to increase the reading achievement of African American students. Studies have shown that rap music, which has its roots in the African American community, can be used as a tool to facilitate this increase, specifically how using rap songs in reading lessons can improve a child's reading motivation, information recall, and vocabulary development. There are also studies on how repeated reading of a text can help improve a child's reading fluency. Yet, there are no studies that combine rap music and repeated reading of a text. This study describes the effects of using a culturally responsive reading strategy on the fluency, decoding, and comprehension skills of African American students. ^ The sample consisted of 105 African American students within eight, second grade classrooms at two different elementary schools. The classes were randomly selected and assigned to the rap group or the control group. Students received eight half-hour sessions using either a rap text or a traditional text in a repeated reading lesson. All of the students were pre-tested and post-tested on the Oral Reading Fluency and the Nonsense Word Fluency portions of the Dynamic Indicators of Basic Literacy Skills Test. Additionally, a researcher constructed comprehension quiz was given to the students at the beginning, middle, and end of the study. Research questions were analyzed using ANOVAs and t tests. ^ The hypotheses were not supported but there was some evidence that rap music in a reading lesson helped improve the fluency skills of African American students at one of the schools. The results also revealed that rap music used in a reading lesson initially improved the comprehension skills of African American students. The rap treatment may not have worked best overall because of the lack of intensity of the treatment. ^ The study has shown some evidence to support using culturally appropriate materials such as rap with students. There needs to be more research on the interaction between teaching methods, materials, and students. ^

The development of direct ultra-fast PCR for forensic genotyping using short channel microfluidic systems with enhanced sieving matrices

There are situations in which it is very important to quickly and positively identify an individual. Examples include suspects detained in the neighborhood of a bombing or terrorist incident, individuals detained attempting to enter or leave the country, and victims of mass disasters. Systems utilized for these purposes must be fast, portable, and easy to maintain. The goal of this project was to develop an ultra fast, direct PCR method for forensic genotyping of oral swabs. The procedure developed eliminates the need for cellular digestion and extraction of the sample by performing those steps in the PCR tube itself. Then, special high-speed polymerases are added which are capable of amplifying a newly developed 7 loci multiplex in under 16 minutes. Following the amplification, a postage stamp sized microfluidic device equipped with specially designed entangled polymer separation matrix, yields a complete genotype in 80 seconds. The entire process is rapid and reliable, reducing the time from sample to genotype from 1-2 days to under 20 minutes. Operation requires minimal equipment and can be easily performed with a small high-speed thermal-cycler, reagents, and a microfluidic device with a laptop. The system was optimized and validated using a number of test parameters and a small test population. The overall precision was better than 0.17 bp and provided a power of discrimination greater than 1 in 106. The small footprint, and ease of use will permit this system to be an effective tool to quickly screen and identify individuals detained at ports of entry, police stations and remote locations. The system is robust, portable and demonstrates to the forensic community a simple solution to the problem of rapid determination of genetic identity.

Rap to Read: Employing a Culturally Relevant Curriculum with African American Students

This study examined the effects of using rap music on the reading skills of 2nd Grade African American students. The sample consisted of 105 students within eight classrooms in two Miami-Dade County public schools. Results revealed that rap was helpful for both fluency and comprehension instruction.

Multiplex PCR for Assessment of Tetracycline Resistance (tet) Genes in Antibiotic-Resistant Soil Bacteria and the Ecological Implications

Antibiotics are becoming increasingly prevalent in bacterial communities due to clinical and agricultural misuse and overuse in their environment. As exposure increases, so does the incidence of microbial resistance. Such is the case with bacterial resistance to tetracyclines, a phenotype often acquired through the horizontal gene transfer of tet genes between bacteria. The objective of this project was to analyze the bacterial diversity of tet resistance genes in soil from Miami-Dade County. Bacterial isolates were Gram-stained and the Kirby-Bauer antibiotic disk diffusion test was performed to determine each bacterium’s degree of resistance. The 16S rRNA gene from antibiotic-resistant isolates was amplified by PCR and sequenced to identify the isolates. All isolates’ tet genes were amplified by multiplex PCR, sequenced, and compared. Among eight isolates, three distinct species were positively identified based on their 16S rRNA sequences and four distinct tet genes were identified, though all tested susceptible to tetracycline via the Kirby-Bauer test. This project clarifies some aspects of the ecology of antibiotic resistance genes, their natural ecological function and the potential for the expansion of intrinsic multi-antibiotic resistance into new ecosystems and/or hosts.

Poéticas do rap engajado e juventudes nas periferias urbanas de Natal-RN

This thesis relates to issues present in the hip hop movement. Thus it includes youngsters grouped in different levels such as local, regional and national. The research deals with this broad segment. The analysis is made on those called “peripheral youth” It encompasses the juvenile segment, that is committed to a movement defined by these youngsters and activists, the so called “hip hop movement”. The research aims to analyze poetics present in rap, considered as an expression of resistance and inventiveness. The analysis was of micro politic nature and it dealt with the “peripheral scenario” present in hip hop Lelo Melodia in the Guarapes neighborhood in Natal-Rio Grande do Norte-Brazil. In this community, the group in known for their resistance attitude. The group´s opinion is not of opposition or force but it is seen in the sense of reinventing life that is currently attached to the persisting social inequality condition. In these terms, the research also deals with this committed poetic rap understood here as expressions towards resistance and inventiveness that is produced by these youth groups that belong to this hip hop movement. The focal group was the Lelo Melodia Group of the Posse de Hip Hop in the Guarapes neighborhood in Natal/Rio Grande do Norte in Brazil.

Leitura do mundo no rap: uma análise bakhtiniana de práticas leitoras no ensino fundamental

This study aims to propose two didactic sequences on the rap songs to the process of formation of critical reflective reader, in the elementary school – in 6th and 9th grade classes. The theoretical foundations that have supported the work are related to the Bakhtin Circle propositions, especially the notions of language as a social ideological discourse; responsive reading; discursive genre and social voices. As for the methodological guidance, it is characterized by adopting the interpretativist paradigm of socio-historical base, in addition to positioning in the large field of Applied Linguistics, whose primary focus is specifically located language. In the selected enunciations to corpus – some Rap songs of protesting – it was analyzed the students’ reflective critical reading in the workshops of Rap-song reading, applied in modules of our teaching sequences. Through the carried out analysis, it was noted that the subjects surveyed have come from an educational culture towards the fulfillment of a conteudista proposed curriculum, so that in this scenario we do not see, through reading, training of the individual citizen, able to through his readings, understanding the socio-ideological structure that surrounds them; and so, escaping of understandings the world, based only on common sense. We conclude also that our teaching proposal represented a novelty for students. Initially, some of them did not see this work as reading, however, the majority was heavily involved in the process; so that, in their comments, we realize voices of a critical and reflective look of which comprise environments, and social situation to which they belong.

Leitura do mundo no rap: uma análise bakhtiniana de práticas leitoras no ensino fundamental

This study aims to propose two didactic sequences on the rap songs to the process of formation of critical reflective reader, in the elementary school – in 6th and 9th grade classes. The theoretical foundations that have supported the work are related to the Bakhtin Circle propositions, especially the notions of language as a social ideological discourse; responsive reading; discursive genre and social voices. As for the methodological guidance, it is characterized by adopting the interpretativist paradigm of socio-historical base, in addition to positioning in the large field of Applied Linguistics, whose primary focus is specifically located language. In the selected enunciations to corpus – some Rap songs of protesting – it was analyzed the students’ reflective critical reading in the workshops of Rap-song reading, applied in modules of our teaching sequences. Through the carried out analysis, it was noted that the subjects surveyed have come from an educational culture towards the fulfillment of a conteudista proposed curriculum, so that in this scenario we do not see, through reading, training of the individual citizen, able to through his readings, understanding the socio-ideological structure that surrounds them; and so, escaping of understandings the world, based only on common sense. We conclude also that our teaching proposal represented a novelty for students. Initially, some of them did not see this work as reading, however, the majority was heavily involved in the process; so that, in their comments, we realize voices of a critical and reflective look of which comprise environments, and social situation to which they belong.

Investigation on performance characteristics of dense graded hot mix asphalt with RAP and crumb rubber

The objective of this study was to fundamentally characterize the laboratory performance of traditional hot mix asphalt (HMA) mixtures incorporating high RAP content and waste tire crumb rubber through their fundamental engineering properties. The nominal maximum aggregates size was chosen for this research was 12mm (considering the limitation of aggregate size for surface layer) and both coarse and fine aggregates are commonly used in Italy that were examined and analyzed in this study. On the other hand, the RAP plays an important role in reducing production costs and enhancing the environmentally sustainable pavements instead of using virgin materials in HMA. Particularly, this study has aimed to use 30% of RAP content (25% fine aggregate RAP and 5% coarse aggregate RAP) and 1% of CR additives by the total weight of aggregates for mix design. The mixture of aggregates, RAP and CR were blended with different amount of unmodified binder through dry processes. Generally, the main purposes of this study were investigating on capability of using RAP and CR in dense graded HMA and comparing the performance of rejuvenator in RAP with CR. In addition, based on the engineering analyses during the study, we were able compare the fundamental Indirect Tensile Strength (ITS) value of dense graded HMA and also mechanical characteristics in terms of Indirect Tensile Stiffness Modulus (ITSM). In order to get an extended comparable data, four groups of different mixtures such as conventional mixture with only virgin aggregates (DV), mixture with RAP (DR), mixture with RAP and rejuvenator (DRR), and mixture with RAP, rejuvenator, CR (DRRCr) were investigated in this research experimentally. Finally, the results of those tests indicated that the mixtures with RAP and CR had the high stiffness and less thermal sensitivity, while the mixture with virgin aggregates only had very low values in comparison.

Aplicación de técnicas de PCR en tiempo real a la trazabilidad y autenticidad de los piensos

La trazabilidad y el correcto etiquetado de los piensos y sus ingredientes son factores esenciales para prevenir fraudes y garantizar la seguridad alimentaria. En el ámbito de la lucha contra las Encefalopatías Espongiformes Transmisibles (EETs), la prohibición de la Unión Europea (UE) de alimentar a rumiantes y otros animales de granja con harinas de carne y huesos derivadas de animales, hace necesaria la disponibilidad de metodologías que permitan identificar el origen de las materias primas e ingredientes presentes en los piensos. El método oficial de análisis microscópico tradicionalmente empleado para este fin presenta limitaciones a la hora de diferenciar entre los huesos de mamíferos y de aves, así como para determinar el origen animal específico de las partículas detectadas. Por ello, una de las prioridades de la UE en los últimos años ha sido potenciar la búsqueda y desarrollo de técnicas analíticas alternativas que permitan la detección específica de todos los componentes que integran los piensos. Teniendo en cuenta estos aspectos, en esta Tesis Doctoral se han desarrollado técnicas de PCR en tiempo real con sondas TaqMan® para el control de autenticidad y trazabilidad de ingredientes de origen animal utilizados en la fabricación de los piensos. Las especies objeto de este trabajo han sido: vaca (Bos taurus), oveja (Ovis aries), cabra (Capra hircos), grupo rumiante, cerdo (Sus scrofa), pollo (Gallus gallos), pavo (Meleagris g-allopavo), pato (Anal platyrhynchos x Cairina moschata), oca (Anser anser), grupo aviar, caballo (Equus caballus), conejo (Oryctolagus cuniculus), liebre (Lepus capensis), grupo lepórido (conejo y liebre) y pescados...

O uso da PCR em tempo real para o estudo da carga parasitária e dos níveis transcricionais durante a infecção experimental por Trypanosoma cruzi

Trypanosoma cruzi is causative agent of Chagas disease, one of most neglected tropical diseases. Estimated that about 11 million people worldwide are infected by T. cruzi and about 6 to 7 million people are at risk in endemic areas. During the process of invasion of host and parasite interact enabling signal transduction and gene expression modulation in response to invasion. The diversity of activated proteins and pathways to repair the damage by disruption of the plasma membrane interest to us and thus present study developed a new form of detection and quantitation by polymerase chain reaction in real time (qPCR) of parasitic load T. cruzi and quantified transcriptional levels relative (RT-qPCR) of dysferlin, Sphingomyelin acid esferase (ASM), transcription factor EB (TFEB) Galectins 1 and 3 and Annexin A2. This study demonstrated that quantification by real time PCR using primers P21fw and P21rv was specific and sensitive for detection of T. cruzi in vivo and in vitro, as well as transcriptional levels of genes related to cytoskeletal organization and repair plasma membrane are modulated in response to damage generated by parasite.

Méthodes moléculaires de détection d'animaux porteurs d'actinobacillus pleuropneumoniae : comparaison et validation d'épreuves PCR

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.