Mechanistic insights aid computational short interfering RNA design.

RNA interference is widely recognized for its utility as a functional genomics tool. In the absence of reliable target site selection tools, however, the impact of RNA interference (RNAi) may be diminished. The primary determinants of silencing are influenced by highly coordinated RNA-protein interactions that occur throughout the RNAi process, including short interfering RNA (siRNA) binding and unwinding followed by target recognition, cleavage, and subsequent product release. Recently developed strategies for identification of functional siRNAs reveal that thermodynamic and siRNA sequence-specific properties are crucial to predict functional duplexes (Khvorova et al., 2003; Reynolds et al., 2004; Schwarz et al., 2003). Additional assessments of siRNA specificity reveal that more sophisticated sequence comparison tools are also required to minimize potential off-target effects (Jackson et al., 2003; Semizarov et al., 2003). This chapter reviews the biological basis for current computational design tools and how best to utilize and assess their predictive capabilities for selecting functional and specific siRNAs.

Oculopharyngeal muscular dystrophy (OPMD): analysis of the PABPN1 gene expansion sequence in 86 patients reveals 13 different expansion types and further evidence for unequal recombination as the mutational mechanism.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant late-onset neuromuscular degenerative disease characterised by proximal muscle weakness, ptosis and swallowing difficulty. The causative genetic abnormality is an expansion consisting of 2-7 additional base triplets in a repeat sequence in exon 1 of the PABPN1 (PABP2) gene and results in an increase in length of the polyalanine tract in the PABPN1 protein from 10 to 12-17 residues. The expansions are stable through meiosis and mitosis suggesting a different mechanism of mutation from that of most other triplet repeat mutations. Most reports describe OPMD expansions as consisting of multiples of a GCG sequence. However, some studies have detected GCA interspersions. We have analysed 86 OPMD patients with a PABPN1 gene expansion, including three compound heterozygotes, and have identified 13 different types of expansion mutation, six of which contain GCA and GCG and almost all of which are consistent with a mutational mechanism of unequal recombination.

Adenovirus type F subtype 41 causing disseminated disease following bone marrow transplantation for immunodeficiency.

Adenovirus causes disseminated disease following bone marrow transplantation (BMT). We report a child who underwent T-cell-depleted BMT. Adenovirus subgenus F serotype 41 was detected antemortem by PCR in cerebrospinal fluid and postmortem in other tissues. Serotypes 40 and 41, associated with gastrointestinal disease, have not previously been implicated in disseminated disease.

Polymerase chain reaction for the laboratory diagnosis of aseptic meningitis and encephalitis

A protocol for testing cerebrospinal fluid specimens using a range of PCR assays for the diagnosis of central nervous system infection was developed and used to test prospectively 383 specimens. PCR assays were used for the detection of adenovirus, Borrelia burgdorferi, enteroviruses, Epstein Barr virus, cytomegalovirus, herpes simplex virus, human herpes virus type 6, JC virus, Leptospira interrogans, Listeria monocytogenes, lymphocytic choriomeningitis virus, measles virus, mumps virus, Mycobacterium sp., Mycoplasma pneumoniae, Toxoplasma gondii and varicella zoster virus. Of the 383 specimens tested in this study, 46 (12.0 percent) were found to be positive. The microorganisms detected were CMV, enterovirus, Epstein Barr virus, herpes simplex virus, human herpes virus type 6, JC virus, L. monocytogenes, Mycobacterium genus, Toxoplasma gondii and varicella zoster virus. The introduction of the PCR protocol described has improved the diagnosis of a range of central nervous system infections in our laboratory. We believe however that further evaluation of these assays in immunocompromised patients is necessary to better determine the predictive value of positive PCR results in these patient groups

Diagnóstico molecular das infecçöes do Sistema Nervoso Central pela Reaçäo em Cadeia da Polimerase (PCR) / Molecular diagnosis of Central Nervous System infections by Polimerase Chain Reaction (PCR)

A identificaçäo do agente etiológico é de fundamental importância no tratamento e prognóstico das infecçöes do Sistema Nervoso Central (SNC) porém, os métodos convencionais de diagnóstico tem limitado sucesso. A Reaçäo em Cadeia da Polimerase (PCR) tem possibilitado o diagnóstico de infecçöes virais,bacterianas e por protozoários de maneira mais rápida e precisa. O objetivo deste estudo foi implantar, em nosso serviço, uma rotina ágil para o diagnóstico molecular dos microorganismos que mais freqüentemente acometem o SNC. As amostras de líquor (LCR) foram testadas para 20 agentes infecciosos: Herpes simplex, Herpes zooster, Enterovírus, vírus da Linfocoriomeningite, vírus Epstein-Barr, Adenovírus, Herpes hominis tipo 6, Sarampo, Caxumba, Mycobacterium sp, Citomegalovírus, Toxoplasma gondii, vírus JC, Listeria monocytogenes, Borrelia burgdorferi, Mycoplasma pneumoniae, Leptospira interrogans, Neisseria meningitidis, Haemophylus influenzae e Streptococcus sp. A técnica usada foi o PCR "nested" com extraçäo do RNA ou DNA das amostras pelo método de Boom modificado. Em algumas reaçöes utilizou-se a combinaçäo de "primers" de patógenos diferentes ("multiplex"), possibilitando a pesquisa de até três microrganismos na mesma reaçäo, agilizando os resultados. Foram testadas 281 amostras de LCR suspeitos de meningite ou meningo-encefalite linfocitária ou asséptica. A PCR foi positiva em 18 amostras (6,4 porcento). microrganismos detectados foram: Mycobacterium sp, Herpes simplex, Citomegalovírus, Herpes hominis tipo 6 e Toxoplasma gondii. Em estudo paralelo com 22 amostras liquóricas de pacientes com meningite bacteriana, a PCR foi positiva em 21 casos (95,4 porcento). Os microrganismos encontrados foram: H. influenzae, Streptococcus sp e N. meningitidis. Em quatro pacientes com PCR positiva a cultura do LCR foi negativa. A introduçäo da técnica de PCR em nosso laboratório otimizou o diagnóstico etiológico das infecçöes do SNC e tem se revelado de grande importância clínica.