Instrumented finger-to-nose test classification in children with ataxia or developmental coordination disorder and controls.

BACKGROUND: During childhood, many conditions may impact coordination. Examples are physiological age-related development and pathological conditions, such as early onset ataxia and developmental coordination disorder. These conditions are generally diagnosed by clinical specialists. However, in absence of a gold phenotypic standard, objective reproducibility among specialists appears limited. METHODS: We investigated whether quantitative analysis of an upper limb coordination task (the finger-to-nose test) could discriminate between physiological and pathological conditions impacting coordination. We used inertial measurement units to estimate movement trajectories of the participants while they executed the finger-to-nose test. We employed random forests to classify each participant in one category. FINDINGS: On average, 87.4% of controls, 74.4% of early onset ataxia and 24.8% of developmental coordination disorder patients were correctly classified. The relatively good classification of early onset ataxia patients and controls contrasts with the poor classification of developmental coordination disorder patients. INTERPRETATION: In absence of a gold phenotypic standard for developmental coordination disorder recognition, it remains elusive whether the finger-to-nose test in these patients represents a sufficiently accurate entity to reflect symptoms distinctive of this disorder. Based on the relatively good results in early onset ataxia patients and controls, we conclude that quantitative analysis of the finger-to-nose test can provide a reliable support tool during the assessment of phenotypic early onset ataxia.

Amplifiable hybridization probes containing a molecular switch.

In order to reduce background signals in Q beta replicase-mediated bioassays, a target-dependent probe amplification strategy has been proposed that utilizes recombinant RNA hybridization probes that contain an inserted molecular switch. A molecular switch is an internal region of the probe that undergoes a conformational change when the probe hybridizes to its target. We investigated whether non-hybridized probes (which cause background signals) could be selectively destroyed by incubating the probe-target hybrids with ribonuclease III, which should cleave the non-hybridized probes and leave the hybridized probes intact. Two problems with this assay design were observed. First, ribonuclease III cleaved probe-target hybrids non-specifically when the target was an RNA, thereby destroying all of the bound probes. And second, the expected conformational change in the molecular switch did not occur when the probes were bound to their targets, apparently because the hairpin stem formed by the molecular switch was too long. Although these results demonstrated that the original assay design could not work, they provided insights that have led to better designs for target-dependent amplification assays. In these assays, the probes will be DNA molecules containing short-stemmed molecular switches. Non-hybridized probes will be selectively destroyed by incubation with a restriction endonuclease.

Quantitative analysis of 16S rDNA using competitive PCR and the QPCR System 5000.

A method for precise and accurate quantification of 16S rDNA has been developed that uses competitive PCR and the QPCR System 5000. The method is based on co-amplification of 16S rDNA sequences, along with an internal standard sequence, using only one set of conserved eubacterial primers. Co-amplified PCR products are rapidly identified and quantified by measuring the electrochemiluminescent signals from specific oligonucleotide reporter probes that are directed against a hypervariable 16S rDNA sequence. Because in the exponential phase of amplification the different target sequences and the internal standard sequence are amplified with the same efficiency, unknown amounts of a target sequence in a sample can be inferred by extrapolating against a standard curve that is generated for the internal standard sequence. This method provides a rapid, nonradioactive and reliable way to simultaneously quantify different specific 16S rDNA targets that are present in low numbers, and may thus be suitable for enumeration of specific target microorganisms in environmental samples.

Comparison of 16S rRNA sequences of segmented filamentous bacteria isolated from mice, rats, and chickens and proposal of "Candidatus Arthromitus".

Segmented filamentous bacteria (SFB) are nonpathogenic bacteria that are commonly found attached to the intestinal walls of many animals. Until now, these bacteria have not been cultured in vitro. Recently, a 16S rRNA sequence analysis revealed that SFB isolated from mice represent a distinct subline within the Clostridium subphylum of the gram-positive bacteria. Since SFB isolated from mice, rats, and chickens are known to be host specific, we investigated the phylogenetic relationships among SFB obtained from these three hosts. Total DNAs from the intestinal floras of chickens and rats were used as templates for PCR amplification of 16S rRNA genes. PCR products were cloned and screened by a dot blot hybridization procedure to identify homologous sequences that cross-reacted with mouse SFB-specific oligonucleotide probes. A phylogenetic analysis of these 16S ribosomal DNA sequences revealed that SFB isolated from these three hosts form a natural group, which is peripherally related to the genus Clostridium sensu stricto (group I Clostridium). The SFB obtained from chickens, rats, and mice had closely related, albeit different, 16S rRNA gene sequences. The observed levels of 16S rRNA sequence divergence, ca. 1.5 to 3%, together with host specificity, suggest that SFB isolated from mice, rats, and chickens represent different species and that coevolution of the SFB and their hosts occurred. "Candidatus Arthromitus" is proposed as the provisional generic name for this group of organisms.